Antibody formulations

ABSTRACT

The present application describes antibody formulations, including monoclonal antibodies formulated in histidine-acetate buffer, as well as a formulation comprising an antibody that binds to domain II of HER2 (for example, Pertuzumab), and a formulation comprising an antibody that binds to DR5 (for example, Apomab).

This application is a continuation of U.S. application Ser. No.16/784,467, filed on Feb. 7, 2020, which is a continuation of U.S.application Ser. No. 15/936,047, filed on Mar. 26, 2018 (now abandoned),which is a continuation of U.S. application Ser. No. 14/672,598, filedon Mar. 30, 2015 (now abandoned), which is a divisional of U.S.application Ser. No. 13/660,114, filed Oct. 25, 2012 (now U.S. Pat. No.9,017,671), which is a divisional application of U.S. application Ser.No. 12/554,194, filed Sep. 4, 2009 (now U.S. Pat. No. 8,372,396), whichis a continuation of to U.S. application Ser. No. 11/254,182 filed Oct.19, 2005 (now abandoned), which claims priority under 35 USC § 119 toU.S. provisional application 60/620,413 filed Oct. 20, 2004, the entiredisclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns antibody formulations, includingmonoclonal antibodies formulated in histidine-acetate buffer, as well asa formulation comprising an antibody that binds to domain II of HER2(for example, Pertuzumab), and a formulation comprising an antibody thatbinds to DR5 (for example, Apomab).

BACKGROUND OF THE INVENTION

In the past ten years, advances in biotechnology have made it possibleto produce a variety of proteins for pharmaceutical applications usingrecombinant DNA techniques. Because proteins are larger and more complexthan traditional organic and inorganic drugs (i.e. possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. For a proteinto remain biologically active, a formulation must preserve intact theconformational integrity of at least a core sequence of the protein'samino acids while at the same time protecting the protein's multiplefunctional groups from degradation. Degradation pathways for proteinscan involve chemical instability (i.e. any process which involvesmodification of the protein by bond formation or cleavage resulting in anew chemical entity) or physical instability (i.e. changes in the higherorder structure of the protein). Chemical instability can result fromdeamidation, racemization, hydrolysis, oxidation, beta elimination ordisulfide exchange. Physical instability can result from denaturation,aggregation, precipitation or adsorption, for example. The three mostcommon protein degradation pathways are protein aggregation, deamidationand oxidation. Cleland et al. Critical Reviews in Therapeutic DrugCarrier Systems 10(4): 307-377 (1993).

Antibody Formulations

Included in the proteins used for pharmaceutical applications areantibodies. An example of an antibody useful for therapy is an antibodywhich binds to the HER2 antigen, such as Pertuzumab.

U.S. Pat. No. 6,339,142 describes a HER2 antibody composition comprisinga mixture of anti-HER2 antibody and one or more acidic variants thereof,wherein the amount of the acidic variant(s) is less than about 25%.Trastuzumab is an exemplified HER2 antibody.

U.S. Pat. Nos. 6,267,958 and 6,685,940 (Andya et al.) describelyophilized antibody formulations, including HER2 and IgE antibodyformulations. WO97/04807 and US 2004/0197326A1 (Fick et al.) describemethods for treating allergic asthma with an IgE antibody. WO99/01556(Lowman et al.) relates to IgE antibody with aspartyl residues prone toisomerization, and improved variants thereof. US 2002/0045571 (Liu etal.) provides reduced viscosity concentrated protein formulations,exemplified by humanized IgE antibody formulations, rhuMAb E25 and E26.WO 02/096457 and US 2004/0170623 (Arvinte et al.) describes stableliquid formulations comprising anti-IgE antibody E25. See, also, US2004/0197324 A1 (Liu and Shire) concerning high concentration anti-IgEformulation.

U.S. Pat. No. 6,171,586 (Lam et al.) describes stable aqueous antibodyformulations. A F(ab′)2 rhuMAb CD18 antibody was formulated in sodiumacetate and histidine-HCl buffers. The preferred formulation for rhuMAbCD18 was 10 mM sodium acetate, 8% trehalose, 0.01% TWEEN 20™, pH 5.0.Acetate (pH 5.0) formulations of rhuMAb CD20 stored at 40. C for onemonth demonstrated greater stability than those samples formulated inhistidine (pH 5.0 or 6.0).

US 2003/0190316 (Kakuta et al.) concerns formulated antibody hPM-1, ahumanized IL-6 receptor antibody. Monomer loss was the greatest insodium citrate (pH 6.7), followed by sodium phosphate (pH 6.8), Tris-HCl(pH 7.2), histidine-HCl (pH 7.2) and glycine (pH 7.6) in descendingorder. The effect of phosphate-Na (pH 6.5), phosphate-His (pH 6.0 or6.5), His-HCl (pH 6.5), and phosphate-Na (pH 6.0) on the stability ofhPM-1 was assessed.

WO 2004/071439 (Burke et al.) state that impurities arose in anatalizumab (anti-alpha4 integrin humanized monoclonal antibody)formulation from the degradation of polysorbate 80, apparently throughan oxidation reaction involving metal ions and hisitidine. Thus, aphosphate buffer was selected.

WO 2000/066160 (English language counterpart EP 1 174 148A1) (Okada etal.) refers to a formulation of a humanized C4G1 antibody which binds toa fibrinogen receptor of a human platelet membrane glycoproteinGPIIb/IIIa, in a sodium phosphate or sodium citrate buffer.

WO 2004/019861 (Johnson et al.) concerns CDP870, a pegylated anti-TNFαFab fragment, formulated at 200 mg/ml in 50 mM sodium acetate (pH 5.5)and 125 mM sodium chloride.

WO 2004/004639 (Nesta, P.) refers to a formulation for huC242-DM1, atumor-activated immunotoxin, in a 50 mM succinic acid buffer (pH 6.0)and sucrose (5% w/v).

WO 03/039485 (Kaisheva et al.) found that Daclizumab (a humanized IL-2receptor antibody) had the highest stability in sodium succinate bufferat pH 6.0, and rapidly lost potency in histidine as the buffer oxidized.

WO 2004/001007 concerns a CD80 monoclonal antibody in a histidine HCl,sodium acetate or sodium citrate buffer.

U.S. Pat. No. 6,252,055 (Relton, J.) refers to anti-CD4 and anti-CD23antibodies formulated in maleate, succinate, sodium acetate or phosphatebuffers, with phosphate being identified as the preferred buffer.

U.S. Pat. No. 5,608,038 (Eibl et al.) refers to highly concentratedpolyclonal immunoglobulin preparations with immunoglobulin, glucose orsucrose, and sodium chloride therein.

WO 03/015894 (Oliver et al.) refers to an aqueous formulation of 100mg/mL SYNAGIS®, 25 mM histidine-HCl, 1.6 mM glycine, pH 6.0, and alyophilized SYNAGIS® which when formulated (before lyophilization)contains 25 mM histidine, 1.6 mM glycine and 3% w/v mannitol at pH 6.0.

US 2004/0191243 A1 (Chen et al.) reports formulation of ABX-IL8, a humanIgG2 antibody.

US 2003/0113316 A1 (Kaisheva et al.) refers to a lyophilized anti-IL2receptor antibody formulation.

HER2 Antibodies

The HER family of receptor tyrosine kinases are important mediators ofcell growth, differentiation and survival. The receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, or HER1), HER2 (ErbB2 or p185^(new)), HER3 (ErbB3) and HER4(ErbB4 or tyro2).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha(TGF-α), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway. Baselga and Mendelsohn Pharmac.Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFRor its ligands, TGF-α and EGF, have been evaluated as therapeutic agentsin the treatment of such malignancies. See, e.g., Baselga andMendelsohn, supra; Masui et al. Cancer Research 44:1002-1007 (1984); andWu et al. J. Clin. Invest. 95:1897-1905 (1995).

The second member of the HER family, p185^(new), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The activated form of the neu proto-oncogeneresults from a point mutation (valine to glutamic acid) in thetransmembrane region of the encoded protein. Amplification of the humanhomolog of neu is observed in breast and ovarian cancers and correlateswith a poor prognosis (Slamon et al., Science, 235:177-182 (1987);Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No.4,968,603). To date, no point mutation analogous to that in the neuproto-oncogene has been reported for human tumors. Overexpression ofHER2 (frequently but not uniformly due to gene amplification) has alsobeen observed in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. See, among others, King et al., Science, 229:974 (1985); Yokotaet al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen etal., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034(1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al.,Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184(1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol.Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363(1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al.,Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate cancer(Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol.28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan etal. J. Urol. 150:126-31 (1993)).

Antibodies directed against the rat p185^(new) and human HER2 proteinproducts have been described. Drebin and colleagues have raisedantibodies against the rat neu gene product, p185^(new) See, forexample, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth.Enzym. 198:277-290 (1991); and WO94/22478. Drebin et al. Oncogene2:273-277(1988) report that mixtures of antibodies reactive with twodistinct regions of p185^(new) result in synergistic anti-tumor effectson neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S.Pat. No. 5,824,311 issued Oct. 20, 1998.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of HER2 antibodies which were characterized usingthe human breast tumor cell line SK-BR-3. Relative cell proliferation ofthe SK-BR-3 cells following exposure to the antibodies was determined bycrystal violet staining of the monolayers after 72 hours. Using thisassay, maximum inhibition was obtained with the antibody called 4D5which inhibited cellular proliferation by 56%. Other antibodies in thepanel reduced cellular proliferation to a lesser extent in this assay.The antibody 4D5 was further found to sensitize HER2-overexpressingbreast tumor cell lines to the cytotoxic effects of TNF-α. See also U.S.Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussedin Hudziak et al. are further characterized in Fendly et al. CancerResearch 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990);Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin.Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol.11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother.37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitettaet al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol.Chem. 269(20):14661-14665 (1994); Scott et al. J Biol. Chem. 266:14300-5(1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994); Lewiset al. Cancer Research 56:1457-1465 (1996); and Schaefer et al. Oncogene15:1385-1394 (1997).

A recombinant humanized version of the murine HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anti-cancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumabreceived marketing approval from the Food and Drug Administration Sep.25, 1998 for the treatment of patients with metastatic breast cancerwhose tumors overexpress the HER2 protein.

Other HER2 antibodies with various properties have been described inTagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancocket al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994);Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

Homology screening has resulted in the identification of two other HERreceptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 aswell as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP PatAppln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA,90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)).Both of these receptors display increased expression on at least somebreast cancer cell lines.

The HER receptors are generally found in various combinations in cellsand heterodimerization is thought to increase the diversity of cellularresponses to a variety of HER ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by sixdifferent ligands; epidermal growth factor (EGF), transforming growthfactor alpha (TGF-α), amphiregulin, heparin binding epidermal growthfactor (HB-EGF), betacellulin and epiregulin (Groenen et al. GrowthFactors 11:235-257 (1994)). A family of heregulin proteins resultingfrom alternative splicing of a single gene are ligands for HER3 andHER4. The heregulin family includes alpha, beta and gamma heregulins(Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869;and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiationfactors (NDFs), glial growth factors (GGFs); acetylcholine receptorinducing activity (ARIA); and sensory and motor neuron derived factor(SMDF). For a review, see Groenen et al. Growth Factors 11:235-257(1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee etal. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligandswere identified; neuregulin-2 (NRG-2) which is reported to bind eitherHER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et alNature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al.PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4(Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin andepiregulin also bind to HER4.

While EGF and TGFα do not bind HER2, EGF stimulates EGFR and HER2 toform a heterodimer, which activates EGFR and results intransphosphorylation of HER2 in the heterodimer. Dimerization and/ortransphosphorylation appears to activate the HER2 tyrosine kinase. SeeEarp et al., supra. Likewise, when HER3 is co-expressed with HER2, anactive signaling complex is formed and antibodies directed against HER2are capable of disrupting this complex (Sliwkowski et al., J. Biol.Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3for heregulin (HRG) is increased to a higher affinity state whenco-expressed with HER2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the HER2-HER3 protein complex. HER4, like HER3, forms anactive signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8(1994)).

To target the HER signaling pathway, rhuMAb 2C4 (Pertuzumab) wasdeveloped as a humanized antibody that inhibits the dimerization of HER2with other HER receptors, thereby inhibiting ligand-drivenphosphorylation and activation, and downstream activation of the RAS andAKT pathways. In a phase I trial of Pertuzumab as a single agent fortreating solid tumors, 3 subjects with advanced ovarian cancer weretreated with Pertuzumab. One had a durable partial response, and anadditional subject had stable disease for 15 weeks Agus et al. Proc AmSoc Clin Oncol 22: 192, Abstract 771 (2003).

DR5 Antibodies

Various ligands and receptors belonging to the tumor necrosis factor(TNF) superfamily have been identified in the art. Included among suchligands are tumor necrosis factor-alpha (“TNF-alpha”), tumor necrosisfactor-beta (“TNF-beta” or “lymphotoxin-alpha”), lymphotoxin-beta(“LT-beta”), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BBligand, LIGHT, Apo-1 ligand (also referred to as Fas ligand or CD95ligand), Apo-2 ligand (also referred to as Apo2L or TRAIL), Apo-3 ligand(also referred to as TWEAK), APRIL, OPG ligand (also referred to as RANKligand, ODF, or TRANCE), and TALL-1 (also referred to as BlyS, BAFF orTHANK) (See, e.g., Ashkenazi, Nature Review, 2:420-430 (2002); Ashkenaziand Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit, Curr.Opin. Cell Biol., 11:255-260 (1999); Golstein, Curr. Biol., 7:R750-R753(1997) Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411;Locksley et al., Cell, 104:487-501 (2001); Gruss and Dower, Blood,85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881(1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987); Pitti et al.,J. Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity,3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993); Armitage etal. Nature, 357:80-82 (1992), WO 97/01633 published Jan. 16, 1997; WO97/25428 published Jul. 17, 1997; Marsters et al., Curr. Biol.,8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410(1997); Hahne et al., J. Exp. Med., 188:1185-1190 (1998); WO98/28426published Jul. 2, 1998; WO98/46751 published Oct. 22, 1998; WO/98/18921published May 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu etal., J Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med.,189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem.,274:15978-15981 (1999)).

Induction of various cellular responses mediated by such TNF familyligands is typically initiated by their binding to specific cellreceptors. Some, but not all, TNF family ligands bind to, and inducevarious biological activity through, cell surface “death receptors” toactivate caspases, or enzymes that carry out the cell death or apoptosispathway (Salvesen et al., Cell, 91:443-446 (1997)). Included among themembers of the TNF receptor superfamily identified to date are TNFR1,TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also referred toas Apo-1 or CD95), DR4 (also referred to as TRAIL-R1), DR5 (alsoreferred to as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG),RANK and Apo-3 (also referred to as DR3 or TRAMP).

Most of these TNF receptor family members share the typical structure ofcell surface receptors including extracellular, transmembrane andintracellular regions, while others are found naturally as solubleproteins lacking a transmembrane and intracellular domain. Theextracellular portion of typical TNFRs contains a repetitive amino acidsequence pattern of multiple cysteine-rich domains (CRDs), starting fromthe NH₂-terminus.

The ligand referred to as Apo-2L or TRAIL was identified several yearsago as a member of the TNF family of cytokines. (see, e.g., Wiley etal., Immunity, 3:673-682 (1995); Pitti et al., J. Biol. Chem.,271:12687-12690 (1996); WO 97/01633; WO 97/25428; U.S. Pat. No.5,763,223 issued Jun. 9, 1998; U.S. Pat. No. 6,284,236 issued Sep. 4,2001). The full-length native sequence human Apo2L/TRAIL polypeptide isa 281 amino acid long, Type II transmembrane protein. Some cells canproduce a natural soluble form of the polypeptide, through enzymaticcleavage of the polypeptide's extracellular region (Mariani et al., J.Cell. Biol., 137:221-229 (1997)). Crystallographic studies of solubleforms of Apo2L/TRAIL reveal a homotrimeric structure similar to thestructures of TNF and other related proteins (Hymowitz et al., Molec.Cell, 4:563-571 (1999); Cha et al., Immunity, 11:253-261 (1999);Mongkolsapaya et al., Nature Structural Biology, 6:1048 (1999); Hymowitzet al., Biochemistry, 39:633-640 (2000)). Apo2L/TRAIL, unlike other TNFfamily members however, was found to have a unique structural feature inthat three cysteine residues (at position 230 of each subunit in thehomotrimer) together coordinate a zinc atom, and that the zinc bindingis important for trimer stability and biological activity. (Hymowitz etal., supra; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)).

It has been reported in the literature that Apo2L/TRAIL may play a rolein immune system modulation, including autoimmune diseases such asrheumatoid arthritis (see, e.g., Thomas et al., J. Immunol.,161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999);Griffith et al., J. Exp. Med., 189:1343-1353 (1999); Song et al., J.Exp. Med., 191:1095-1103 (2000)).

Soluble forms of Apo2L/TRAIL have also been reported to induce apoptosisin a variety of cancer cells, including colon, lung, breast, prostate,bladder, kidney, ovarian and brain tumors, as well as melanoma,leukemia, and multiple myeloma (see, e.g., Wiley et al., supra; Pitti etal., supra; U.S. Pat. No. 6,030,945 issued Feb. 29, 2000; U.S. Pat. No.6,746,668 issued Jun. 8, 2004; Rieger et al., FEBS Letters, 427:124-128(1998); Ashkenazi et al., J. Clin. Invest., 104:155-162 (1999); Walczaket al., Nature Med., 5:157-163 (1999); Keane et al., Cancer Research,59:734-741 (1999); Mizutani et al., Clin. Cancer Res., 5:2605-2612(1999); Gazitt, Leukemia, 13:1817-1824 (1999); Yu et al., Cancer Res.,60:2384-2389 (2000); Chinnaiyan et al., Proc. Natl. Acad. Sci.,97:1754-1759 (2000)). In vivo studies in murine tumor models furthersuggest that Apo2L/TRAIL, alone or in combination with chemotherapy orradiation therapy, can exert substantial anti-tumor effects (see, e.g.,Ashkenazi et al., supra; Walzcak et al., supra; Gliniak et al., CancerRes., 59:6153-6158 (1999); Chinnaiyan et al., supra; Roth et al.,Biochem. Biophys. Res. Comm., 265:479-483 (1999); PCT ApplicationUS/00/15512; PCT Application US/01/23691). In contrast to many types ofcancer cells, most normal human cell types appear to be resistant toapoptosis induction by certain recombinant forms of Apo2L/TRAIL(Ashkenazi et al., supra; Walzcak et al., supra). Jo et al. has reportedthat a polyhistidine-tagged soluble form of Apo2L/TRAIL inducedapoptosis in vitro in normal isolated human, but not non-human,hepatocytes (Jo et al., Nature Med., 6:564-567 (2000); see also, Nagata,Nature Med., 6:502-503 (2000)). It is believed that certain recombinantApo2L/TRAIL preparations may vary in terms of biochemical properties andbiological activities on diseased versus normal cells, depending, forexample, on the presence or absence of a tag molecule, zinc content, and% trimer content (See, Lawrence et al., Nature Med., Letter to theEditor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor,7:385-386 (2001)).

Apo2L/TRAIL has been found to bind at least five different receptors. Atleast two of the receptors which bind Apo2L/TRAIL contain a functional,cytoplasmic death domain. One such receptor has been referred to as“DR4” (and alternatively as TR4 or TRAIL-R1) (Pan et al., Science,276:111-113 (1997); see also WO98/32856 published Jul. 30, 1998;WO99/37684 published Jul. 29, 1999; WO 00/73349 published Dec. 7, 2000;U.S. Pat. No. 6,433,147 issued Aug. 13, 2002; U.S. Pat. No. 6,461,823issued Oct. 8, 2002, and U.S. Pat. No. 6,342,383 issued Jan. 29, 2002).

Another such receptor for Apo2L/TRAIL has been referred to as DR5 (ithas also been alternatively referred to as Apo-2; TRAIL-R or TRAIL-R2,TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see, e.g., Sheridan et al.,Science, 277:818-821 (1997), Pan et al., Science, 277:815-818 (1997),WO98/51793 published Nov. 19, 1998; WO98/41629 published Sep. 24, 1998;Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J.,16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997);WO98/35986 published Aug. 20, 1998; EP870,827 published Oct. 14, 1998;WO98/46643 published Oct. 22, 1998; WO99/02653 published Jan. 21, 1999;WO99/09165 published Feb. 25, 1999; WO99/11791 published Mar. 11, 1999;US 2002/0072091 published Aug. 13, 2002; US 2002/0098550 published Dec.7, 2001; U.S. Pat. No. 6,313,269 issued Dec. 6, 2001; US 2001/0010924published Aug. 2, 2001; US 2003/0125540 published Jul. 3, 2003; US2002/0160446 published Oct. 31, 2002, US 2002/0048785 published Apr. 25,2002; U.S. Pat. No. 6,342,369 issued February, 2002; U.S. Pat. No.6,569,642 issued May 27, 2003, U.S. Pat. No. 6,072,047 issued Jun. 6,2000, U.S. Pat. No. 6,642,358 issued Nov. 4, 2003; IS 6,743,625 issuedJun. 1, 2004). Like DR4, DR5 is reported to contain a cytoplasmic deathdomain and be capable of signaling apoptosis upon ligand binding (orupon binding a molecule, such as an agonist antibody, which mimics theactivity of the ligand). The crystal structure of the complex formedbetween Apo-2L/TRAIL and DR5 is described in Hymowitz et al., MolecularCell, 4:563-571 (1999).

Upon ligand binding, both DR4 and DR5 can trigger apoptosisindependently by recruiting and activating the apoptosis initiator,caspase-8, through the death-domain-containing adaptor molecule referredto as FADD/Mort1 (Kischkel et al., Immunity, 12:611-620 (2000); Spricket al., Immunity, 12:599-609 (2000); Bodmer et al., Nature Cell Biol.,2:241-243 (2000)).

Apo2L/TRAIL has been reported to also bind those receptors referred toas DcR1, DcR2 and OPG, which believed to function as inhibitors, ratherthan transducers of signaling (see., e.g., DcR1 (also referred to asTRID, LIT or TRAIL-R3) (Pan et al., Science, 276:111-113 (1997);Sheridan et al., Science, 277:818-821 (1997); MacFarlane et al., J.Biol. Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters,416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170(1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)); DcR2(also called TRUNDD or TRAIL-R4) (Marsters et al., Curr. Biol.,7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);Degli-Esposti et al., Immunity, 7:813-820 (1997)), and OPG. In contrastto DR4 and DR5, the DcR1 and DcR2 receptors do not signal apoptosis.

Certain antibodies which bind to the DR4 and/or DR5 receptors have beenreported in the literature. For example, anti-DR4 antibodies directed tothe DR4 receptor and having agonistic or apoptotic activity in certainmammalian cells are described in, e.g., WO 99/37684 published Jul. 29,1999; WO 00/73349 published Jul. 12, 2000; WO 03/066661 published Aug.14, 2003. See, also, e.g., Griffith et al., J. Immunol., 162:2597-2605(1999); Chuntharapai et al., J. Immunol., 166:4891-4898 (2001); WO02/097033 published Dec. 2, 2002; WO 03/042367 published May 22, 2003;WO 03/038043 published May 8, 2003; WO 03/037913 published May 8, 2003.Certain anti-DR5 antibodies have likewise been described, see, e.g., WO98/51793 published Nov. 8, 1998; Griffith et al., J. Immunol.,162:2597-2605 (1999); Ichikawa et al., Nature Med., 7:954-960 (2001);Hylander et al., “An Antibody to DR5 (TRAIL-Receptor 2) Suppresses theGrowth of Patient Derived Gastrointestinal Tumors Grown in SCID mice”,Abstract, 2d International Congress on Monoclonal Antibodies in Cancers,Aug. 29-Sep. 1, 2002, Banff, Alberta, Canada; WO 03/038043 published May8, 2003; WO 03/037913 published May 8, 2003. In addition, certainantibodies having cross-reactivity to both DR4 and DR5 receptors havebeen described (see, e.g., U.S. Pat. No. 6,252,050 issued Jun. 26,2001).

SUMMARY OF THE INVENTION

The invention herein relates, at least in part, to the identification ofhistidine-acetate, pH 5.5 to 6.5, as a particularly useful buffer forformulating monoclonal antibodies, especially full length IgG1antibodies which are susceptible to deamidation and/or aggregation. Theformulation retards degradation of the antibody product therein.

Thus, in a first aspect, the invention concerns a stable pharmaceuticalformulation comprising a monoclonal antibody in histidine-acetatebuffer, pH 5.5 to 6.5. The monoclonal antibody preferably binds anantigen selected from the group consisting of HER2, CD20, DR5, BR3, IgE,and VEGF.

In addition, the invention concerns a method of treating a disease ordisorder in a subject comprising administering the formulation to asubject in an amount effective to treat the disease or disorder.

In another aspect, the invention concerns a pharmaceutical formulationcomprising: (a) a full length IgG1 antibody susceptible to deamidationor aggregation in an amount from about 10 mg/mL to about 250 mg/mL; (b)histidine-acetate buffer, pH 5.5 to 6.5; (c) saccharide selected fromthe group consisting of trehalose and sucrose, in an amount from about60 mM to about 250 mM; and (d) polysorbate 20 in an amount from about0.01% to about 0.1%.

The invention also provides a method for reducing deamidation oraggregation of a therapeutic monoclonal antibody, comprising formulatingthe antibody in a histidine-acetate buffer, pH 5.5 to 6.5.

In yet a further aspect, the invention concerns a pharmaceuticalformulation comprising an antibody that binds to domain II of HER2 in ahistidine buffer at a pH from about 5.5 to about 6.5, a saccharide and asurfactant.

The invention also relates to a pharmaceutical formulation comprisingPertuzumab in an amount from about 20 mg/mL to about 40 mg/mL,histidine-acetate buffer, sucrose, and polysorbate 20, wherein the pH ofthe formulation is from about 5.5 to about 6.5.

The invention also pertains to a pharmaceutical formulation comprising aDR5 antibody in a histidine buffer at a pH from about 5.5 to about 6.5,a saccharide, and a surfactant.

In another aspect, the invention concerns a pharmaceutical formulationcomprising Apomab in an amount from about 10 mg/mL to about 30 mg/mL,histidine-acetate buffer, trehalose, and polysorbate 20, wherein the pHof the formulation is from about 5.5 to about 6.5.

In yet another aspect, the invention provides a method of treatingcancer in a subject, comprising administering the pharmaceuticalformulation to the subject in an amount effective to treat the cancer.

The invention also concerns a vial with a stopper pierceable by asyringe or a stainless steel tank comprising the formulation inside thevial or tank, optionally in frozen form.

Moreover, the invention provides a method of making a pharmaceuticalformulation comprising: (a) preparing the monoclonal antibodyformulation; and (b) evaluating physical stability, chemical stability,or biological activity of the monoclonal antibody in the formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Domains I-IV (SEQ ID Nos.19-22, respectively) of theextracellular domain of HER2.

FIGS. 2A and 2B depict alignments of the amino acid sequences of thevariable light (V_(L)) (FIG. 2A) and variable heavy (V_(H)) (FIG. 2B)domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 1 and 2,respectively); V_(L) and V_(H) domains of humanized 2C4 version 574 (SEQID Nos. 3 and 4, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III)(SEQ ID Nos. 5 and 6, respectively). Asterisks identify differencesbetween humanized 2C4 version 574 and murine monoclonal antibody 2C4 orbetween humanized 2C4 version 574 and the human framework.Complementarity Determining Regions (CDRs) are in brackets.

FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chainand heavy chain (SEQ ID Nos. 15 and 16, respectively). CDRs are shown inbold. Calculated molecular mass of the light chain and heavy chain are23,526.22 Da and 49,216.56 Da (cysteines in reduced form). Thecarbohydrate moiety is attached to Asn 299 of the heavy chain.

FIGS. 4A and 4B show the amino acid sequences of Pertuzumab light andheavy chain, each including an intact amino terminal signal peptidesequence (SEQ ID Nos. 17 and 18, respectively).

FIG. 5 depicts, schematically, binding of 2C4 at the heterodimericbinding site of HER2, thereby preventing heterodimerization withactivated EGFR or HER3.

FIG. 6 depicts coupling of HER2/HER3 to the MAPK and Akt pathways.

FIG. 7 compares activities of Trastuzumab and Pertuzumab.

FIG. 8 depicts stability of Pertuzumab formulation by ion exchange (IEX)analyses.

FIG. 9 shows stability of Pertuzumab formulation by size exclusionchromatography (SEC) analysis.

FIG. 10 reflects physical stability Pertuzumab in differentformulations.

FIG. 11 is from an agitation study of Pertuzumab liquid formulations.

FIG. 12 is from another agitation study of Pertuzumab liquidformulations.

FIG. 13 is from a freeze-thawing study of Pertuzumab formulation.

FIGS. 14A and 14B show the amino acid sequences of Trastuzumab lightchain (SEQ ID No. 13) and heavy chain (SEQ ID No. 14).

FIGS. 15A and 15B depict a variant Pertuzumab light chain sequence (SEQID No. 23) and a variant Pertuzumab heavy chain sequence (SEQ ID No.24).

FIGS. 16A and 16B show oligosaccharide structures commonly observed inIgG antibodies.

FIGS. 17A and 17B show the sequences of the light and heavy chains (SEQID Nos. 37-44) of specific anti-IgE antibodies E25, E26, HAE1 andHu-901. In FIG. 17A, the variable light domain ends with the residuesVEIK, residue 111. In FIG. 17B, the variable heavy domain ends with theresidues VTVSS, around residue 120.

FIG. 18A is a sequence alignment comparing the amino acid sequences ofthe variable light domain (V_(L)) of each of murine 2H7 (SEQ ID No. 25),humanized 2H7v16 variant (SEQ ID No. 26), and the human kappa lightchain subgroup I (SEQ ID No. 27). The CDRs of V_(L) of 2H7 and hu2H7v16are as follows: CDR1 (SEQ ID No. 57), CDR2 (SEQ ID No. 58), and CDR3(SEQ ID No. 59).

FIG. 18B is a sequence alignment comparing the amino acid sequences ofthe variable heavy domain (V_(H)) of each of murine 2H7 (SEQ ID No. 28),humanized 2H7v16 variant (SEQ ID No. 29), and the human consensussequence of the heavy chain subgroup III (SEQ ID No. 30). The CDRs ofV_(H) of 2H7 and hu2H7v16 are as follows: CDR1 (SEQ ID No. 60), CDR2(SEQ ID No. 61), and CDR3 (SEQ ID No. 62).

In FIG. 18A and FIG. 18B, the CDR1, CDR2 and CDR3 in each chain areenclosed within brackets, flanked by the framework regions, FR1-FR4, asindicated. 2H7 refers to murine 2H7 antibody. The asterisks in betweentwo rows of sequences indicate the positions that are different betweenthe two sequences. Residue numbering is according to Kabat et al.Sequences of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), with insertionsshown as a, b, c, d, and e.

FIG. 19 depicts variable domain sequences of three different VEGFantibodies with SEQ ID Nos. 31-36.

FIG. 20 shows size exclusion chromatography (SEC) elution profile of thefollowing Apomab samples: (a) control and formulations prepared at (b)pH 4.0, (c) pH 5.0, (d) pH 6.0 and (e) pH 7.0. The formulated sampleswere stored at 40° C. for 2 months prior to the analysis.

FIG. 21 depicts pH rate profile for the loss in Apomab antibody monomerduring storage. Monomer kinetics by SEC was monitored during storage at30° C. and 40° C. and the first-order rate constants were calculated.

FIG. 22 provides ion exchange chromatography (IEC) elution profile ofApomab samples as follows: (a) control and formulations prepared at (b)pH 4.0, (c) pH 5.0, (d) pH 6.0 and (e) pH 7.0. The formulated sampleswere stored at 40° C. for 2 months prior to the analysis.

FIG. 23 shows pH rate profile for the loss in IEC main peak duringstorage. Main peak kinetics by IEC was monitored during storage at 30°C. and 40° C. and the first-order rate constants were calculated.

FIG. 24 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ IDNo. 45) and its derived amino acid sequence (SEQ ID No. 46). The “N” atnucleotide position 447 (in SEQ ID No. 45) is used to indicate thenucleotide base may be a “T” or “G”.

FIGS. 25A and 25B show the 411 amino acid sequence of human DR5 receptor(SEQ ID No. 47) as published in WO 98/51793 on Nov. 19, 1998, and theencoding nucleotide sequence (SEQ ID No. 48).

FIGS. 26A and 26B show the 440 amino acid sequence of human DR5 receptor(SEQ ID No. 49) and the encoding nucleotide sequence (SEQ ID No. 50), asalso published in WO 98/35986 on Aug. 20, 1998.

FIG. 27 shows the Apomab 7.3 heavy chain amino acid sequence (SEQ ID No.51).

FIG. 28 shows the Apomab 7.3 light chain amino acid sequence (SEQ ID No.52).

FIG. 29 shows the alignment of 16E2 heavy chain (SEQ ID No. 53) andApomab 7.3 heavy chain (SEQ ID No. 51) amino acid sequences.

FIG. 30 shows the alignment of 16E2 light chain (SEQ ID No. 54) andApomab 7.3 light chain (SEQ ID No. 52) amino acid sequences.

FIGS. 31A and 31B depict the variable heavy amino acid sequence (FIG.31A; SEQ ID No. 55) and variable light amino acid sequence (FIG. 31B;SEQ ID No. 56) of Apomab 7.3. CDR residues are identified in bold.

FIG. 32 shows an alignment of the mature 2H7v16 and 2H7v511 light chains(SEQ ID Nos. 63 and 64, respectively). Sequences shown with Kabatvariable domain residue numbering and Eu constant domain residuenumbering.

FIG. 33 shows an alignment of the mature 2H7v16 and 2H7v511 heavy chains(SEQ ID Nos. 65 and 66, respectively). Sequences shown with Kabatvariable domain residue numbering and Eu constant domain residuenumbering.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

A “sterile” formulation is asceptic or free from all livingmicroorganisms and their spores.

Herein, a “frozen” formulation is one at a temperature below 0° C.Generally, the frozen formulation is not freeze-dried, nor is itsubjected to prior, or subsequent, lyophilization. Preferably, thefrozen formulation comprises frozen drug substance for storage (instainless steel tank) or frozen drug product (in final vialconfiguration).

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Preferably, the formulation is stable at about 40° C. for atleast about 2-4 weeks, and/or stable at about 5° C. and/or 15° C. for atleast 3 months, and/or stable at about −20° C. for at least 3 months orat least 1 year. Furthermore, the formulation is preferably stablefollowing freezing (to, e.g., −70° C.) and thawing of the formulation,for example following 1, 2 or 3 cycles of freezing and thawing.Stability can be evaluated qualitatively and/or quantitatively in avariety of different ways, including evaluation of aggregate formation(for example using size exclusion chromatography, by measuringturbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography or capillary zoneelectrophoresis; amino-terminal or carboxy-terminal sequence analysis;mass spectrometric analysis; SDS-PAGE analysis to compare reduced andintact antibody; peptide map (for example tryptic or LYS-C) analysis;evaluating biological activity or antigen binding function of theantibody; etc. Instability may involve any one or more of: aggregation,deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation),isomerization (e.g. Asp isomeriation), clipping/hydrolysis/fragmentation(e.g. hinge region fragmentation), succinimide formation, unpairedcysteine(s), N-terminal extension, C-terminal processing, glycosylationdifferences, etc.

A “deamidated” monoclonal antibody herein is one in which one or moreasparagine residue thereof has been derivitized, e.g. to an asparticacid or an iso-aspartic acid.

An antibody which is “susceptible to deamidation” is one comprising oneor more residue which has been found to be prone to deamidate.

An antibody which is “susceptible to aggregation” is one which has beenfound to aggregate with other antibody molecule(s), especially uponfreezing and/or agitation.

An antibody which is “susceptible to fragmentation” is one which hasbeen found to be cleaved into two or more fragments, for example at ahinge region thereof.

By “reducing deamidation, aggregation, or fragmentation” is intendedpreventing or decreasing the amount of deamidation, aggregation, orfragmentation relative to the monoclonal antibody formulated at adifferent pH or in a different buffer.

Herein, “biological activity” of a monoclonal antibody refers to theability of the antibody to bind to antigen and result in a measurablebiological response which can be measured in vitro or in vivo. Suchactivity may be antagonistic (for example where the antibody is a HER2antibody) or agonistic (for instance where the antibody binds DR5). Inthe case of Pertuzumab, in one embodiment, the biological activityrefers to the ability of the formulated antibody to inhibitproliferation of the human breast cancer cell line MDA-MB-175-VII. Wherethe antibody is Apomab, the biological activity can refer, for example,to the ability of the formulated antibody to kill colon carcinoma,Colo205, cells.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention preferably has a pH in the range from about 5.0to about 7.0, preferably from about 5.5 to about 6.5, for example fromabout 5.8 to about 6.2, and most preferably has a pH of about 6.0.Examples of buffers that will control the pH in this range includeacetate, succinate, succinate, gluconate, histidine, citrate,glycylglycine and other organic acid buffers. The preferred bufferherein is a histidine buffer.

A “histidine buffer” is a buffer comprising histidine ions. Examples ofhistidine buffers include histidine chloride, histidine acetate,histidine phosphate, histidine sulfate. The preferred histidine bufferidentified in the examples herein was found to be histidine acetate. Inthe preferred embodiment, the histidine acetate buffer is prepared bytitrating L-histidine (free base, solid) with acetic acid (liquid).Preferably, the histidine buffer or histidine-acetate buffer is at pH5.5 to 6.5, preferably pH 5.8 to 6.2.

A “saccharide” herein comprises the general composition (CH2O)n andderivatives thereof, including monosaccharides, disaccharides,trisaccharides, polysaccharides, sugar alcohols, reducing sugars,nonreducing sugars, etc. Examples of saccharides herein include glucose,sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin,dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol,mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose,lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc.The preferred saccharide herein is a nonreducing disaccharide, such astrehalose or sucrose.

Herein, a “surfactant” refers to a surface-active agent, preferably anonionic surfactant. Examples of surfactants herein include polysorbate(for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g.poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. Thepreferred surfactant herein is polysorbate 20.

A “HER receptor” is a receptor protein tyrosine kinase which belongs tothe HER receptor family and includes EGFR, HER2, HER3 and HER4 receptorsand other members of this family to be identified in the future. The HERreceptor will generally comprise an extracellular domain, which may bindan HER ligand; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.Preferably the HER receptor is native sequence human HER receptor.

The extracellular domain of HER2 comprises four domains, Domain I (aminoacid residues from about 1-195), Domain II (amino acid residues fromabout 196-320), Domain III (amino acid residues from about 321-488), andDomain IV (amino acid residues from about 489-632) (residue numberingwithout signal peptide). See Garrett et al. Mol. Cell. 11: 495-505(2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. CancerCell 5:317-328 (2004), or Plowman et al. Proc. Natl. Acad. Sci.90:1746-1750 (1993). See also FIG. 1 herein.

The terms “ErbB1,” “HER1”, “epidermal growth factor receptor” and “EGFR”are used interchangeably herein and refer to EGFR as disclosed, forexample, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987),including naturally occurring mutant forms thereof (e.g. a deletionmutant EGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1refers to the gene encoding the EGFR protein product.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234(1986) (Genebank accession number X03363). The term “erbB2” refers tothe gene encoding human ErbB2 and “neu” refers to the gene encoding ratp185^(new). Preferred HER2 is native sequence human HER2.

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989).

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appln No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including isoforms thereof, e.g., asdisclosed in WO99/19488, published Apr. 22, 1999.

By “HER ligand” is meant a polypeptide which binds to and/or activates aHER receptor. The HER ligand of particular interest herein is a nativesequence human HER ligand such as epidermal growth factor (EGF) (Savageet al., J. Biol. Chem. 247:7612-7621 (1972)); transforming growth factoralpha (TGF-α) (Marquardt et al., Science 223:1079-1082 (1984));amphiregulin also known as schwanoma or keratinocyte autocrine growthfactor (Shoyab et al. Science 243:1074-1076 (1989); Kimura et al. Nature348:257-260 (1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557(1991)); betacellulin (Shing et al., Science 259:1604-1607 (1993); andSasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al.,Science 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem.270:7495-7500 (1995); and Komurasaki et al. Oncogene 15:2841-2848(1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,Nature 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc.Natl. Acad. Sci. 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari etal. Oncogene 18:2681-89 (1999)) or cripto (CR-1) (Kannan et al. J. Biol.Chem. 272(6):3330-3335 (1997)). HER ligands which bind EGFR include EGF,TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligandswhich bind HER3 include heregulins. HER ligands capable of binding HER4include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 andheregulins.

“Heregulin” (HRG) when used herein refers to a polypeptide encoded bythe heregulin gene product as disclosed in U.S. Pat. No. 5,641,869 orMarchionni et al., Nature, 362:312-318 (1993). Examples of heregulinsinclude heregulin-α, heregulin-β1, heregulin-β2 and heregulin-β3 (Holmeset al., Science, 256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neudifferentiation factor (NDF) (Peles et al. Cell 69: 205-216 (1992));acetylcholine receptor-inducing activity (ARIA) (Falls et al. Cell72:801-815 (1993)); glial growth factors (GGFs) (Marchionni et al.,Nature, 362:312-318 (1993)); sensory and motor neuron derived factor(SMDF) (Ho et al. J. Biol. Chem. 270:14523-14532 (1995)); γ-heregulin(Schaefer et al. Oncogene 15:1385-1394 (1997)). The term includesbiologically active fragments and/or amino acid sequence variants of anative sequence HRG polypeptide, such as an EGF-like domain fragmentthereof (e.g. HRGβ1₁₇₇₋₂₄₄).

A “HER dimer” herein is a noncovalently associated dimer comprising atleast two different HER receptors. Such complexes may form when a cellexpressing two or more HER receptors is exposed to an HER ligand and canbe isolated by immunoprecipitation and analyzed by SDS-PAGE as describedin Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), forexample. Examples of such HER dimers include EGFR-HER2, HER2-HER3 andHER3-HER4 heterodimers. Moreover, the HER dimer may comprise two or moreHER2 receptors combined with a different HER receptor, such as HER3,HER4 or EGFR. Other proteins, such as a cytokine receptor subunit (e.g.gp130) may be associated with the dimer.

A “heterodimeric binding site” on HER2, refers to a region in theextracellular domain of HER2 that contacts, or interfaces with, a regionin the extracellular domain of EGFR, HER3 or HER4 upon formation of adimer therewith. The region is found in Domain II of HER2. Franklin etal. Cancer Cell 5:317-328 (2004).

“HER activation” or “HER2 activation” refers to activation, orphosphorylation, of any one or more HER receptors, or HER2 receptors.Generally, HER activation results in signal transduction (e.g. thatcaused by an intracellular kinase domain of a HER receptorphosphorylating tyrosine residues in the HER receptor or a substratepolypeptide). HER activation may be mediated by HER ligand binding to aHER dimer comprising the HER receptor of interest. HER ligand binding toa HER dimer may activate a kinase domain of one or more of the HERreceptors in the dimer and thereby results in phosphorylation oftyrosine residues in one or more of the HER receptors and/orphosphorylation of tyrosine residues in additional substratepolypeptides(s), such as Akt or MAPK intracellular kinases.

The term “antibody” herein is used in the broadest sense andspecifically covers full length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two full length antibodies, and antibody fragments, solong as they exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences.

“Antibody fragments” comprise a portion of a full length antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s).

A “full length antibody” is one which comprises an antigen-bindingvariable region as well as a light chain constant domain (CL) and heavychain constant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domainsmay be native sequence constant domains (e.g. human native sequenceconstant domains) or amino acid sequence variants thereof. Preferably,the full length antibody has one or more effector functions.

The term “main species antibody” herein refers to the antibody structurein a composition which is the quantitatively predominant antibodymolecule in the composition. In one embodiment, the main speciesantibody is a HER2 antibody, such as an antibody that binds to Domain IIof HER2, antibody that inhibits HER dimerization more effectively thanTrastuzumab, and/or an antibody which binds to a heterodimeric bindingsite of HER2. The preferred embodiment herein of a main species HER2antibody is one comprising the variable light and variable heavy aminoacid sequences in SEQ ID Nos. 3 and 4, and most preferably comprisingthe light chain and heavy chain amino acid sequences in SEQ ID Nos. 15and 16 (Pertuzumab).

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about 70%homology with the main species antibody, and preferably, they will be atleast about 80%, more preferably at least about 90% homologous with themain species antibody. The amino acid sequence variants possesssubstitutions, deletions, and/or additions at certain positions withinor adjacent to the amino acid sequence of the main species antibody.Examples of amino acid sequence variants herein include acidic variant(e.g. deamidated antibody variant), basic variant, the antibody with anamino-terminal leader extension (e.g. VHS-) on one or two light chainsthereof, antibody with a C-terminal lysine residue on one or two heavychains thereof, etc, and includes combinations of variations to theamino acid sequences of heavy and/or light chains. The antibody variantof particular interest herein is the antibody comprising anamino-terminal leader extension on one or two light chains thereof,optionally further comprising other amino acid sequence and/orglycosylation differences relative to the main species antibody.

A “therapeutic monoclonal antibody” is an antibody used for therapy of ahuman subject. Therapeutic monoclonal antibodies disclosed hereininclude: HER2 antibodies for cancer and various non-malignant diseasesor disorders; CD20 or BR3 antibodies for therapy of B cell malignancies,autoimmune diseases, graft rejection, or blocking an immune response toa foreign antigen; IgE antibodies for therapy of an IgE-mediateddisorder; DR5 or VEGF antibodies for cancer therapy.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moeities attached thereto which differ from one ormore carbohydrate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead a GO oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations.

Where the antibody has an Fc region, an oligosaccharide structure suchas that shown in FIG. 16 herein may be attached to one or two heavychains of the antibody, e.g. at residue 299 (298, Eu numbering ofresidues). For Pertuzumab, G0 was the predominant oligosaccharidestructure, with other oligosaccharide structures such as GO-F, G-1,Man5, Man6, G1-1, G1(1-6), G1(1-3) and G2 being found in lesser amountsin the Pertuzumab composition.

Unless indicated otherwise, a “G1 oligosaccharide structure” hereinincludes G-1, G1-1, G1(1-6) and G1(1-3) structures.

An “amino-terminal leader extension” herein refers to one or more aminoacid residues of the amino-terminal leader sequence that are present atthe amino-terminus of any one or more heavy or light chains of anantibody. An exemplary amino-terminal leader extension comprises orconsists of three amino acid residues, VHS, present on one or both lightchains of an antibody variant.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and Fcγ RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain C_(H)1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear at least one free thiol group. F(ab′)₂ antibodyfragments originally were produced as pairs of Fab′ fragments which havehinge cysteines between them. Other chemical couplings of antibodyfragments are also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). HER2 antibody scFv fragments are described in WO93/16185; U.S.Pat. Nos. 5,571,894; and 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 orTrastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No.5,821,337 expressly incorporated herein by reference; humanized 520C9(WO93/21319) and humanized 2C4 antibodies as described herein.

For the purposes herein, “Trastuzumab,” “HERCEPTIN®,” and “huMAb4D5-8”refer to an antibody comprising the light and heavy chain amino acidsequences in SEQ ID NOS. 13 and 14, respectively.

Herein, “Pertuzumab” and “rhuMAb 2C4” refer to an antibody comprisingthe variable light and variable heavy amino acid sequences in SEQ IDNos. 3 and 4, respectfully. Where Pertuzumab is a full length antibody,it preferably comprises the light chain and heavy chain amino acidsequences in SEQ ID NOS. 15 and 16, respectively.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety orradiolabel.

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by V_(H) and V_(L) domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004(1995); Jackson et al., J Immunol. 154(7):3310-9 (1995); and Hawkins etal, J. Mol. Biol. 226:889-896 (1992).

An “agonist antibody” is an antibody which binds to and activates areceptor. Generally, the receptor activation capability of the agonistantibody will be at least qualitatively similar (and may be essentiallyquantitatively similar) to a native agonist ligand of the receptor. Anexample of an agonist antibody is one which binds to a receptor in theTNF receptor superfamily, such as DR5, and induces apoptosis of cellsexpressing the TNF receptor (e.g. DR5). Assays for determining inductionof apoptosis are described in WO98/51793 and WO99/37684, both of whichare expressly incorporated herein by reference.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

A HER2 antibody which “inhibits HER dimerization more effectively thanTrastuzumab” is one which reduces or eliminates HER dimers moreeffectively (for example at least about 2-fold more effectively) thanTrastuzumab. Preferably, such an antibody inhibits HER2 dimerization atleast about as effectively as an antibody selected from the groupconsisting of murine monoclonal antibody 2C4, a Fab fragment of murinemonoclonal antibody 2C4, Pertuzumab, and a Fab fragment of Pertuzumab.One can evaluate HER dimerization inhibition by studying HER dimersdirectly, or by evaluating HER activation, or downstream signaling,which results from HER dimerization, and/or by evaluating theantibody-HER2 binding site, etc. Assays for screening for antibodieswith the ability to inhibit HER dimerization more effectively thanTrastuzumab are described in Agus et al. Cancer Cell 2: 127-137 (2002)and WO01/00245 (Adams et al.). By way of example only, one may assay forinhibition of HER dimerization by assessing, for example, inhibition ofHER dimer formation (see, e.g., FIG. 1A-B of Agus et al. Cancer Cell 2:127-137 (2002); and WO01/00245); reduction in HER ligand activation ofcells which express HER dimers (WO01/00245 and FIG. 2A-B of Agus et al.Cancer Cell 2: 127-137 (2002), for example); blocking of HER ligandbinding to cells which express HER dimers (WO01/00245, and FIG. 2E ofAgus et al. Cancer Cell 2: 127-137 (2002), for example); cell growthinhibition of cancer cells (e.g. MCF7, MDA-MD-134, ZR-75-1, MD-MB-175,T-47D cells) which express HER dimers in the presence (or absence) ofHER ligand (WO01/00245 and FIGS. 3A-D of Agus et al. Cancer Cell 2:127-137 (2002), for instance); inhibition of downstream signaling (forinstance, inhibition of HRG-dependent AKT phosphorylation or inhibitionof HRG- or TGFα-dependent MAPK phosphorylation) (see, WO01/00245, andFIG. 2C-D of Agus et al. Cancer Cell 2: 127-137 (2002), for example).One may also assess whether the antibody inhibits HER dimerization bystudying the antibody-HER2 binding site, for instance, by evaluating astructure or model, such as a crystal structure, of the antibody boundto HER2 (See, for example, Franklin et al. Cancer Cell 5:317-328(2004)).

The HER2 antibody may “inhibit HRG-dependent AKT phosphorylation” and/orinhibit “HRG- or TGFα-dependent MAPK phosphorylation” more effectively(for instance at least 2-fold more effectively) than Trastuzumab (seeAgus et al. Cancer Cell 2: 127-137 (2002) and WO01/00245, by way ofexample).

The HER2 antibody may be one which does “not inhibit HER2 ectodomaincleavage” (Molina et al. Cancer Res. 61:4744-4749(2001).

A HER2 antibody that “binds to a heterodimeric binding site” of HER2,binds to residues in domain II (and optionally also binds to residues inother of the domains of the HER2 extracellular domain, such as domains Iand III), and can sterically hinder, at least to some extent, formationof a HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimer. Franklin et al.Cancer Cell 5:317-328 (2004) characterize the HER2-Pertuzumab crystalstructure, deposited with the RCSB Protein Data Bank (ID Code IS78),illustrating an exemplary antibody that binds to the heterodimericbinding site of HER2.

An antibody that “binds to domain II” of HER2 binds to residues indomain II and optionally residues in other domain(s) of HER2, such asdomains I and III. Preferably the antibody that binds to domain II bindsto the junction between domains I, II and III of HER2.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a HER expressingcancer cell either in vitro or in vivo. Thus, the growth inhibitoryagent may be one which significantly reduces the percentage of HERexpressing cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. Examples of “growth inhibitory”antibodies are those which bind to HER2 and inhibit the growth of cancercells overexpressing HER2. Preferred growth inhibitory HER2 antibodiesinhibit growth of SK-BR-3 breast tumor cells in cell culture by greaterthan 20%, and preferably greater than 50% (e.g. from about 50% to about100%) at an antibody concentration of about 0.5 to 30 pg/ml, where thegrowth inhibition is determined six days after exposure of the SK-BR-3cells to the antibody (see U.S. Pat. No. 5,677,171 issued Oct. 14,1997). The SK-BR-3 cell growth inhibition assay is described in moredetail in that patent and hereinbelow. The preferred growth inhibitoryantibody is a humanized variant of murine monoclonal antibody 4D5, e.g.,Trastuzumab.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which expresses the antigen to which the antibodybinds. Preferably the cell is a tumor cell. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using cells that express an antigen to which the antibodybinds. Examples of antibodies that induce apoptosis are HER2 antibodies7C2 and 7F3, and certain DR5 antibodies.

The “epitope 2C4” is the region in the extracellular domain of HER2 towhich the antibody 2C4 binds. In order to screen for antibodies whichbind to the 2C4 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprisesresidues from domain II in the extracellular domain of HER2. 2C4 andPertuzumab bind to the extracellular domain of HER2 at the junction ofdomains I, II and III. Franklin et al. Cancer Cell 5:317-328 (2004).

The “epitope 4D5” is the region in the extracellular domain of HER2 towhich the antibody 4D5 (ATCC CRL 10463) and Trastuzumab bind. Thisepitope is close to the transmembrane domain of HER2, and within DomainIV of HER2. To screen for antibodies which bind to the 4D5 epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Alternatively, epitope mapping can beperformed to assess whether the antibody binds to the 4D5 epitope ofHER2 (e.g. any one or more residues in the region from about residue 529to about residue 625, inclusive, of HER2).

The “epitope 7C2/7F3” is the region at the amino terminus, within DomainI, of the extracellular domain of HER2 to which the 7C2 and/or 7F3antibodies (each deposited with the ATCC, see below) bind. To screen forantibodies which bind to the 7C2/7F3 epitope, a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Alternatively, epitope mapping can be performed to establishwhether the antibody binds to the 7C2/7F3 epitope on HER2 (e.g. any oneor more of residues in the region from about residue 22 to about residue53 of HER2).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease is to beprevented. Hence, the patient to be treated herein may have beendiagnosed as having the disease or may be predisposed or susceptible tothe disease.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophagael cancer, tumors of the biliary tract, as well as head and neckcancer.

The term “effective amount” refers to an amount of a drug effective to adisease in the patient. Where the disease is cancer, the effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. To the extent the drug may preventgrowth and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. The effective amount may extend progression free survival,result in an objective response (including a partial response, PR, orcomplete response, CR), increase overall survival time, and/or improveone or more symptoms of cancer.

A “HER2-expressing cancer” is one comprising cells which have HER2protein present at their cell surface.

A cancer which “overexpresses” a HER receptor is one which hassignificantly higher levels of a HER receptor, such as HER2, at the cellsurface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. HER receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the HER protein present on the surface of a cell(e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of HER-encoding nucleic acid in thecell, e.g. via fluorescent in situ hybridization (FISH; see WO98/45479published October, 1998), southern blotting, or polymerase chainreaction (PCR) techniques, such as real time quantitative PCR (RT-PCR).One may also study HER receptor overexpression by measuring shed antigen(e.g., HER extracellular domain) in a biological fluid such as serum(see, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995;and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside from theabove assays, various in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g. a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g. by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

Conversely, a cancer which “does not overexpress HER2 receptor” is onewhich does not express higher than normal levels of HER2 receptorcompared to a noncancerous cell of the same tissue type.

A cancer which “overexpresses” a HER ligand is one which producessignificantly higher levels of that ligand compared to a noncancerouscell of the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation.Overexpression of the HER ligand may be determined diagnostically byevaluating levels of the ligand (or nucleic acid encoding it) in thepatient, e.g. in a tumor biopsy or by various diagnostic assays such asthe IHC, FISH, southern blotting, PCR or in vivo assays described above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm⁵³, Bi²¹², P³² and radioactive isotopesof Lu), chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegalI (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®),liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomaldoxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate,gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), anepothilone, and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfornithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin, andcarboplatin; vincas, which prevent tubulin polymerization from formingmicrotubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®),vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®);etoposide (VP-16); ifosfamide; mitoxantrone; leucovovin; novantrone;edatrexate; daunomycin; aminopterin; ibandronate; topoisomeraseinhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such asretinoic acid, including bexarotene (TARGRETIN®); bisphosphonates suchas clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®),NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine,COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor(e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577);orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium(GENASENSE®); pixantrone; EGFR inhibitors (see definition below);tyrosine kinase inhibitors (see definition below); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone, and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens withmixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®),4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene,raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogenreceptor modulators (SERMs) such as SERM3; pure anti-estrogens withoutagonist properties, such as fulvestrant (FASLODEX®), and EM800 (suchagents may block estrogen receptor (ER) dimerization, inhibit DNAbinding, increase ER turnover, and/or suppress ER levels); aromataseinhibitors, including steroidal aromatase inhibitors such as formestaneand exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors suchas anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide,and other aromatase inhibitors including vorozole (RIVISOR®), megestrolacetate (MEGASE®), fadrozole, imidazole; lutenizing hormone-releasinghormone agonists, including leuprolide (LUPRON® and ELIGARD®),goserelin, buserelin, and tripterelin; sex steroids, includingprogestines such as megestrol acetate and medroxyprogesterone acetate,estrogens such as diethylstilbestrol and premarin, andandrogens/retinoids such as fluoxymesterone, all transretionic acid andfenretinide; onapristone; anti-progesterones; estrogen receptordown-regulators (ERDs); anti-androgens such as flutamide, nilutamide andbicalutamide; testolactone; and pharmaceutically acceptable salts, acidsor derivatives of any of the above; as well as combinations of two ormore of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF (see WO98/50433, Abgenix). The anti-EGFR antibodymay be conjugated with a cytotoxic agent, thus generating animmunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). Examples ofsmall molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSA™;Astra Zeneca), CP-358774 or Erlotinib HCL (TARCEVA™; Genentech/OSI) andAG1478, AG1571 (SU 5271; Sugen).

A “tyrosine kinase inhibitor” is a molecule which inhibits to someextent tyrosine kinase activity of a tyrosine kinase such as a HERreceptor. Examples of such inhibitors include the EGFR-targeted drugsnoted in the preceding paragraph as well as small molecule HER2 tyrosinekinase inhibitor such as TAK165 available from Takeda, dual-HERinhibitors such as EKB-569 (available from Wyeth) which preferentiallybinds EGFR but inhibits both HER2 & EGFR-overexpressing cells, GW572016(available from Glaxo) an oral HER2 and EGFR tyrosine kinase inhibitor,and PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibits Raf-1signaling; non-HER targeted TK inhibitors such as Imatinib mesylate(Gleevac™) available from Glaxo; MAPK extracellular regulated kinase Iinhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines;pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophenemoieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g. thosethat bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No.5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such asCI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate(Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or asdescribed in any of the following patent publications: U.S. Pat. No.5,804,396; WO99/09016 (American Cyanimid); WO98/43960 (AmericanCyanamid); WO97/38983 (Warner Lambert); WO99/06378 (Warner Lambert);WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc); WO96/33978(Zeneca); WO96/3397 (Zeneca); and WO96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The preferred anti-angiogenic factorherein is an antibody that binds to Vascular Endothelial Growth Factor(VEGF), such as Bevacizumab (AVASTIN®).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The antibody which is formulated is preferably essentially pure anddesirably essentially homogeneous (i.e. free from contaminating proteinsetc). “Essentially pure” antibody means a composition comprising atleast about 90% by weight of the antibody, based on total weight of thecomposition, preferably at least about 95% by weight. “Essentiallyhomogeneous” antibody means a composition comprising at least about 99%by weight of antibody, based on total weight of the composition.

A “B-cell surface marker” or “B-cell surface antigen” herein is anantigen expressed on the surface of a B cell that can be targeted withan antibody that binds thereto. Exemplary B-cell surface markers includethe CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72,CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82,CD83, CDw84, CD85 and CD86 leukocyte surface markers (for descriptions,see The Leukocyte Antigen Facts Book, 2^(nd) Edition. 1997, ed. Barclayet al. Academic Press, Harcourt Brace & Co., New York). Other B-cellsurface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB,CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3,IRTA1, FcRH6, BCMA, and 239287. The B-cell surface marker of particularinterest herein is preferentially expressed on B cells compared to othernon-B-cell tissues of a mammal and may be expressed on both precursor Bcells and mature B cells. The preferred B-cell surface marker herein isCD20 or BR3.

The “CD20” antigen, or “CD20,” is an about 35-kDa, non-glycosylatedphosphoprotein found on the surface of greater than 90% of B cells fromperipheral blood or lymphoid organs. CD20 is present on both normal Bcells as well as malignant B cells, but is not expressed on stem cells.Other names for CD20 in the literature include “B-lymphocyte-restrictedantigen” and “Bp35”. The CD20 antigen is described in Clark et al. Proc.Natl. Acad. Sci. (USA) 82:1766 (1985), for example.

Purely for the purposes herein, “humanized 2H7” refers to a humanizedvariant of the 2H7 antibody whose CDR sequences are disclosed in U.S.Pat. No. 5,500,362 (FIGS. 5 and 6 ), expressly incorporated herein byreference. Examples of humanized 2H7 antibodies herein include thevariants described in WO2004/056312, also expressly incorporated hereinby reference, as well as other variants, including, but not limited to:2H7v16, 2H7v31, 2H7v73, 2H7v75, 2H7v96, 2H7v114, 2H7v115, 2H7v116,2H7v138, 2H7v477, 2H7v375, etc.

In one embodiment, the humanized 2H7 antibody comprises one, two, three,four, five or six of the following CDR sequences:

CDR L1 sequence RASSSVSYXH wherein X is M or L (SEQ ID No. 67), forexample SEQ ID No. 57 (FIG. 18A),CDR L2 sequence of SEQ ID No. 58 (FIG. 18A),CDR L3 sequence QQWXFNPPT wherein X is S or A (SEQ ID No. 68), forexample SEQ ID No. 59 (FIG. 18A),CDR H1 sequence of SEQ ID No. 60 (FIG. 18B),CDR H2 sequence of AIYPGNGXTSYNQKFKG wherein X is D or A (SEQ ID No.69), for example SEQ ID No. 61 (FIG. 18B), andCDR H3 sequence of VVYYSXXYWYFDV wherein the X at position 6 is N, A, Y,W or D, and the X at position 7 is S or R (SEQ ID No. 70), for exampleSEQ ID No. 62 (FIG. 18B).

The CDR sequences above are generally present within human variablelight and variable heavy framework sequences, such as substantially thehuman consensus FR residues of human light chain kappa subgroup I(V_(L)6I), and substantially the human consensus FR residues of humanheavy chain subgroup III (V_(H)III). See also WO 2004/056312 (Lowman etal.).

The variable heavy region may be joined to a human IgG chain constantregion, wherein the region may be, for example, IgG1 or IgG3, includingnative sequence and variant constant regions.

In a preferred embodiment, such antibody comprises the variable heavydomain sequence of SEQ ID No. 29 (v16, as shown in FIG. 18B), optionallyalso comprising the variable light domain sequence of SEQ ID No. 26(v16, as shown in FIG. 18A), which optionally comprises one or moreamino acid substitution(s) at positions 56, 100, and/or 100a, e.g. D56A,N100A or N100Y, and/or S100aR in the variable heavy domain and one ormore amino acid substitution(s) at positions 32 and/or 92, e.g. M32Land/or S92A, in the variable light domain. Preferably, the antibody isan intact antibody comprising the light chain amino acid sequences ofSEQ ID Nos. 63 or 64, and heavy chain amino acid sequences of SEQ ID No.65, 66, 71 or 72.

A preferred humanized 2H7 antibody is ocrelizumab (Genentech).

The antibody herein may further comprise at least one amino acidsubstitution in the Fc region that improves ADCC activity, such as onewherein the amino acid substitutions are at positions 298, 333, and 334,preferably S298A, E333A, and K334A, using Eu numbering of heavy chainresidues. See also U.S. Pat. No. 6,737,056B1, Presta.

Any of these antibodies may comprise at least one substitution in the Fcregion that improves FcRn binding or serum half-life, for example asubstitution at heavy chain position 434, such as N434W. See also U.S.Pat. No. 6,737,056B1, Presta.

Any of these antibodies may further comprise at least one amino acidsubstitution in the Fc region that increases CDC activity, for example,comprising at least a substitution at position 326, preferably K326A orK326W. See also U.S. Pat. No. 6,528,624B1 (Idusogie et al.).

Some preferred humanized 2H7 variants are those comprising the variablelight domain of SEQ ID No. 26 and the variable heavy domain of SEQ IDNo. 29, including those with or without substitutions in an Fc region(if present), and those comprising a variable heavy domain withalteration N100A; or D56A and N100A; or D56A, N100Y, and S100aR; in SEQID No. 29 and a variable light domain with alteration M32L; or S92A; orM32L and S92A; in SEQ ID No. 26.

M34 in the variable heavy chain of 2H7v16 has been identified as apotential source of antibody stability and is another potentialcandidate for substitution.

In a summary of some various preferred embodiments of the invention, thevariable region of variants based on 2H7v16 comprise the amino acidsequences of v16 except at the positions of amino acid substitutionsthat are indicated in the Table below. Unless otherwise indicated, the2H7 variants will have the same light chain as that of v16.

Exemplary Humanized 2H7 Antibody Variants

2H7 Heavy chain Light chain Version (V_(H)) changes (V_(L)) changes Fcchanges 16 for — reference 31 — — S298A, E333A, K334A 73 N100A M32L 75N100A M32L S298A, E333A, K334A 96 D56A, N100A S92A 114 D56A, N100A M32L,S92A S298A, E333A, K334A 115 D56A, N100A M32L, S92A S298A, E333A, K334A,E356D, M358L 116 D56A, N100A M32L, S92A S298A, K334A, K322A 138 D56A,N100A M32L, S92A S298A, E333A, K334A, K326A 477 D56A, N100A M32L, S92AS298A, E333A, K334A, K326A, N434W 375 — — K334L 588 — — S298A, E333A,K334A, K326A 511 D56A. N100Y, M32L, S92A S298A, E333A, K334A, S100aRK326A

One preferred humanized 2H7 comprises 2H7v16 variable light domainsequence:

(SEQ ID No. 26) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG TKVEIKR;

and 2H7v16 variable heavy domain sequence:

(SEQ ID No. 29) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS.

Where the humanized 2H7v16 antibody is an intact antibody, it maycomprise the light chain amino acid sequence:

(SEQ ID No. 63) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC;

and the heavy chain amino acid sequence of SEQ ID No. 65 or:

(SEQ ID No. 71) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.

Another preferred humanized 2H7 antibody comprises 2H7v511 variablelight domain sequence:

(SEQ ID No. 73) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG TKVEIKR

and 2H7v511 variable heavy domain sequence:

(SEQ ID No. 74) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS.

Where the humanized 2H7v511 antibody is an intact antibody, it maycomprise the light chain amino acid sequence:

(SEQ ID No. 64) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECand the heavy chain amino acid sequence of SEQ ID No. 66 or:

(SEQ ID No. 72) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.

A “B-cell malignancy” herein includes non-Hodgkin's lymphoma (NHL),including low grade/follicular NHL, small lymphocytic (SL) NHL,intermediate grade/follicular NHL, intermediate grade diffuse NHL, highgrade immunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia; leukemia,including acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), Hairy cell leukemia and chronic myeloblastic leukemia;and other hematologic malignancies. Such malignancies may be treatedwith antibodies directed against B-cell surface markers, such as CD20.

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology, ThirdEdition; A. Victor Hoffbrand and John E. Pettit (eds.) (HarcourtPublishers Limited 2000) (see, in particular FIG. 11.57, 11.58 and/or11.59). More specific examples include, but are not limited to, relapsedor refractory NHL, front line low grade NHL, Stage III/IV NHL,chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/orlymphoma, small lymphocytic lymphoma, B cell chronic lymphacyticleukemia and/or prolymphocytic leukemia and/or small lymphocyticlymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/orlymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone—MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) T-celllymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma and/orleukemia, T cell chronic lymphocytic leukemia and/or prolymphacyticleukemia, large granular lymphocytic leukemia, mycosis fungoides and/orSezary syndrome, extranodal natural killer/T-cell (nasal type) lymphoma,enteropathy type T-cell lymphoma, hepatosplenic T-cell lymphoma,subcutaneous panniculitis like T-cell lymphoma, skin (cutaneous)lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma,intestinal T cell lymphoma, peripheral T-cell (not otherwise specified)lymphoma and angioimmunoblastic T-cell lymphoma.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis, juvenile-onset rheumatoid arthritis,osteoarthritis, psoriatic arthritis, and ankylosing spondylitis),psoriasis, dermatitis including atopic dermatitis, chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, toxic epidermal necrolysis, scleroderma(including systemic scleroderma), sclerosis such as progressive systemicsclerosis, inflammatory bowel disease (IBD) (for example, Crohn'sdisease, ulcerative colitis, autoimmune inflammatory bowel disease),pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,episcleritis), respiratory distress syndrome, including adultrespiratory distress syndrome (ARDS), meningitis, IgE-mediated diseasessuch as anaphylaxis and allergic and atopic rhinitis, encephalitis suchas Rasmussen's encephalitis, uveitis or autoimmune uveitis, colitis suchas microscopic colitis and collagenous colitis, glomerulonephritis (GN)such as membranous GN (membranous nephropathy), idiopathic membranousGN, membranous proliferative GN (MPGN), including Type I and Type II,and rapidly progressive GN, allergic conditions, allergic reaction,eczema, asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) suchas cutaneous SLE, subacute cutaneous lupus erythematosus, lupus(including nephritis, cerebritis, pediatric, non-renal, discoid,alopecia), juvenile onset (Type I) diabetes mellitus, includingpediatric insulin-dependent diabetes mellitus (IDDM), adult onsetdiabetes mellitus (Type II diabetes), multiple sclerosis (MS) such asspino-optical MS, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including lymphomatoid granulomatosis,Wegener's granulomatosis, agranulocytosis, vasculitis (including largevessel vasculitis (including polymyalgia rheumatica and giant cell(Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki'sdisease and polyarteritis nodosa), CNS vasculitis, systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia,Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia orimmune hemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency,hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseasesinvolving leukocyte diapedesis, CNS inflammatory disorders, multipleorgan injury syndrome, antigen-antibody complex mediated diseases,anti-glomerular basement membrane disease, anti-phospholipid antibodysyndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman'ssyndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren'ssyndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoidbullous, pemphigus (including vulgaris, foliaceus, and pemphigusmucus-membrane pemphigoid), autoimmune polyendocrinopathies, Reiter'sdisease, immune complex nephritis, chronic neuropathy such as IgMpolyneuropathies or IgM-mediated neuropathy, thrombocytopenia (asdeveloped by myocardial infarction patients, for example), includingthrombotic thrombocytopenic purpura (TTP) and autoimmune orimmune-mediated thrombocytopenia such as idiopathic thrombocytopenicpurpura (ITP) including chronic or acute ITP, autoimmune disease of thetestis and ovary including autoimmune orchitis and oophoritis, primaryhypothyroidism, hypoparathyroidism, autoimmune endocrine diseasesincluding thyroiditis such as autoimmune thyroiditis, chronicthyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis,autoimmune thyroid disease, idiopathic hypothyroidism, Addison'sdisease, Grave's disease, polyglandular syndromes such as autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),paraneoplastic syndromes, including neurologic paraneoplastic syndromessuch as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome,stiff-man or stiff-person syndrome, encephalomyelitis such as allergicencephalomyelitis, myasthenia gravis, cerebellar degeneration, limbicand/or brainstem encephalitis, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, Sheehan's syndrome,autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, chronicactive hepatitis or autoimmune chronic active hepatitis, lymphoidinterstitial pneumonitis, bronchiolitis obliterans (non-transplant) vsNSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy),primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractorysprue, dermatitis herpetiformis, cryoglobulinemia, amylotrophic lateralsclerosis (ALS; Lou Gehrig's disease), coronary artery disease,autoimmune inner ear disease (AIED) or autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractorypolychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cellhepatitis, scleritis, a non-cancerous lymphocytosis, a primarylymphocytosis, which includes monoclonal B cell lymphocytosis (e.g.,benign monoclonal gammopathy and monoclonal garnmopathy of undeterminedsignificance, MGUS), peripheral neuropathy, paraneoplastic syndrome,channelopathies such as epilepsy, migraine, arrhythmia, musculardisorders, deafness, blindness, periodic paralysis, and channelopathiesof the CNS, autism, inflammatory myopathy, focal segmentalglomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis,autoimmune hepatological disorder, fibromyalgia, multiple endocrinefailure, Schmidt's syndrome, adrenalitis, gastric atrophy, preseniledementia, demyelinating diseases, Dressler's syndrome, alopecia arcata,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyly, and telangiectasia), male and femaleautoimmune infertility, ankylosing spondolytis, mixed connective tissuedisease, Chagas' disease, rheumatic fever, recurrent abortion, farmer'slung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome,bird-fancier's lung, Alport's syndrome, alveolitis such as allergicalveolitis and fibrosing alveolitis, interstitial lung disease,transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis,schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan'ssyndrome, dengue, endocarditis, endomyocardial fibrosis,endophthalmitis, erythema elevatum et diutinum, erythroblastosisfetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome,flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer'sdisease, parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, and giant cell polymyalgia.

The “tumor necrosis factor receptor superfamily” or “TNF receptorsuperfamily” herein refers to receptor polypeptides bound by cytokinesin the TNF family. Generally, these receptors are Type I transmembranereceptors with one or more cysteine rich repeat sequences in theirextracellular domain. The TNF receptor superfamily may be furthersubdivided into (1) death receptors; (2) decoy receptors; and (3)signaling receptors that lack death domains. The “death receptors”contain in their cytoplasmic or intracellular region a “death domain”,i.e., a region or sequence which acts to transduce signals in the cellwhich can result in apoptosis or in induction of certain genes. The“decoy receptors” lack a functional death domain and are incapable oftransducing signals which result in apoptosis. Examples of cytokines inthe TNF gene family include Tumor Necrosis Factor-alpha (TNF-alpha),Tumor Necrosis Factor-beta (TNF-beta or lymphotoxin), CD30 ligand, CD27ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (alsoreferred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referredto as TRAIL), Apo-3 ligand (also referred to as TWEAK), osteoprotegerin(OPG), APRIL, RANK ligand (also referred to as TRANCE), and TALL-1 (alsoreferred to as BlyS, BAFF or THANK). Examples of receptors in the TNFreceptor superfamily include: type 1 Tumor Necrosis Factor Receptor(TNFR1), type 2 Tumor Necrosis Factor Receptor (TNFR2), p75 Nerve GrowthFactor receptor (NGFR), the B cell surface antigen CD40, the T cellantigen OX-40, Apo-1 receptor (also called Fas or CD95), Apo-3 receptor(also called DR3, swl-1, TRAMP and LARD), the receptor called“Transmembrane Activator and CAML-Interactor” or “TACI”, BCMA protein,DR4, DR5 (alternatively referred to as Apo-2; TRAIL-R2, TR6, Tango-63,hAPO8, TRICK2 or KILLER), DR6, DcR1 (also referred to as TRID, LIT orTRAIL-R3), DcR2 (also called TRAIL-R4 or TRUNDD), OPG, DcR3 (also calledTR6 or M68), CAR1, HVEM (also called ATAR or TR2), GITR, ZTNFR-5, NTR-1,TNFL1, CD30, Lymphotoxin beta receptor (LTBr), 4-1BB receptor and TR9(EP988, 371A1).

The terms “Apo-2 ligand”, “Apo-2L”, “Apo2L”, Apo-2 ligand/TRAIL” and“TRAIL” are used herein interchangeably to refer to a polypeptidesequence which includes amino acid residues 114-281, inclusive, 95-281,inclusive, residues 92-281, inclusive, residues 91-281, inclusive,residues 41-281, inclusive, residues 39-281, inclusive, residues 15-281,inclusive, or residues 1-281, inclusive, of the amino acid sequenceshown in FIG. 24 (SEQ ID No. 46), as well as biologically activefragments, deletional, insertional, and/or substitutional variants ofthe above sequences. In one embodiment, the polypeptide sequencecomprises residues 114-281 of FIG. 24 (SEQ ID No. 46). Optionally, thepolypeptide sequence comprises residues 92-281 or residues 91-281 ofFIG. 24 (SEQ ID No. 46). The Apo-2L polypeptides may be encoded by thenative nucleotide sequence shown in FIG. 24 (SEQ ID No. 45). Optionally,the codon which encodes residue Pro119 (FIG. 24 ; SEQ ID No. 45) may be“CCT” or “CCG”. Optionally, the fragments or variants are biologicallyactive and have at least about 80% amino acid sequence identity, or atleast about 90% sequence identity, or at least 95%, 96%, 97%, 98%, or99% sequence identity with any one of the above sequences. Thedefinition encompasses substitutional variants of Apo-2 ligand in whichat least one of its native amino acids are substituted by another aminoacid such as an alanine residue. The definition also encompasses anative sequence Apo-2 ligand isolated from an Apo-2 ligand source orprepared by recombinant and/or synthetic methods. The Apo-2 ligand ofthe invention includes the polypeptides referred to as Apo-2 ligand orTRAIL disclosed in WO97/01633 published Jan. 16, 1997, WO97/25428published Jul. 17, 1997, WO99/36535 published Jul. 22, 1999, WO 01/00832published Jan. 4, 2001, WO02/09755 published Feb. 7, 2002, WO 00/75191published Dec. 14, 2000, and U.S. Pat. No. 6,030,945 issued Feb. 29,2000. The terms are used to refer generally to forms of the Apo-2 ligandwhich include monomer, dimer, trimer, hexamer or height oligomer formsof the polypeptide. All numbering of amino acid residues referred to inthe Apo-2L sequence use the numbering according to FIG. 24 (SEQ ID No.46), unless specifically stated otherwise.

“Apo-2 ligand receptor” includes the receptors referred to in the art as“DR4” and “DR5.” Pan et al. have described the TNF receptor familymember referred to as “DR4” (Pan et al., Science, 276:111-113 (1997);see also WO98/32856 published Jul. 30, 1998; WO 99/37684 published Jul.29, 1999; WO 00/73349 published Dec. 7, 2000; U.S. Pat. No. 6,433,147issued Aug. 13, 2002; U.S. Pat. No. 6,461,823 issued Oct. 8, 2002, andU.S. Pat. No. 6,342,383 issued Jan. 29, 2002). Sheridan et al., Science,277:818-821 (1997) and Pan et al., Science, 277:815-818 (1997) describedanother receptor for Apo2L/TRAIL (see also, WO98/51793 published Nov.19, 1998; WO98/41629 published Sep. 24, 1998). This receptor is referredto as DR5 (the receptor has also been alternatively referred to asApo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al.,Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387(1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO98/35986published Aug. 20, 1998; EP870,827 published Oct. 14, 1998; WO98/46643published Oct. 22, 1998; WO99/02653 published Jan. 21, 1999; WO99/09165published Feb. 25, 1999; WO99/11791 published Mar. 11, 1999; US2002/0072091 published Aug. 13, 2002; US 2002/0098550 published Dec. 7,2001; U.S. Pat. No. 6,313,269 issued Dec. 6, 2001; US 2001/0010924published Aug. 2, 2001; US 2003/01255540 published Jul. 3, 2003; US2002/0160446 published Oct. 31, 2002, US 2002/0048785 published Apr. 25,2002; U.S. Pat. No. 6,569,642 issued May 27, 2003, U.S. Pat. No.6,072,047 issued Jun. 6, 2000, U.S. Pat. No. 6,642,358 issued Nov. 4,2003). As described above, other receptors for Apo-2L include DcR1,DcR2, and OPG. The term “Apo-2L receptor” when used herein encompassesnative sequence receptor and receptor variants. These terms encompassApo-2L receptor expressed in a variety of mammals, including humans.Apo-2L receptor may be endogenously expressed as occurs naturally in avariety of human tissue lineages, or may be expressed by recombinant orsynthetic methods. A “native sequence Apo-2L receptor” comprises apolypeptide having the same amino acid sequence as an Apo-2L receptorderived from nature. Thus, a native sequence Apo-2L receptor can havethe amino acid sequence of naturally-occurring Apo-2L receptor from anymammal, including humans. Such native sequence Apo-2L receptor can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence Apo-2L receptor” specificallyencompasses naturally-occurring truncated or secreted forms of thereceptor (e.g., a soluble form containing, for instance, anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants.Receptor variants may include fragments or deletion mutants of thenative sequence Apo-2L receptor. FIGS. 25A-C show the 411 amino acidsequence of human DR5 receptor, along with its nucleotide sequence (SEQID Nos. 47 and 48) as published in WO 98/51793 on Nov. 19, 1998. Atranscriptional splice variant of human DR5 receptor is known in theart. This splice variant encodes the 440 amino acid sequence of humanDR5 receptor as shown in FIGS. 26A-C, along with its nucleotide sequence(SEQ ID Nos. 49 and 50), and as published in WO 98/35986 on Aug. 20,1998.

“Death receptor antibody” is used herein to refer generally to antibodyor antibodies directed to a receptor in the tumor necrosis factorreceptor superfamily and containing a death domain capable of signallingapoptosis, and such antibodies include DR5 antibody and DR4 antibody.

“DR5 receptor antibody”, “DR5 antibody”, or “anti-DR5 antibody” is usedin a broad sense to refer to antibodies that bind to at least one formof a DR5 receptor or extracellular domain thereof. Optionally the DR5antibody is fused or linked to a heterologous sequence or molecule.Preferably the heterologous sequence allows or assists the antibody toform higher order or oligomeric complexes. Optionally, the DR5 antibodybinds to DR5 receptor but does not bind or cross-react with anyadditional Apo-2L receptor (e.g. DR4, DcR1, or DcR2). Optionally theantibody is an agonist of DR5 signalling activity.

Optionally, the DR5 antibody of the invention binds to a DR5 receptor ata concentration range of about 0.1 nM to about 20 mM as measured in aBIAcore binding assay. Optionally, the DR5 antibodies of the inventionexhibit an IC50 value of about 0.6 nM to about 18 mM as measured in aBIAcore binding assay.

Purely for the purposes herein, the term “Apomab” refers to an agonistantibody which binds to DR5 and comprises the variable heavy andvariable light amino acid sequences of SEQ ID Nos. 55 and 56. PreferablyApomab comprises the heavy and light chains of SEQ ID Nos. 51 and 52,respectively.

II. Production of Antibodies

Techniques for producing antibodies which can be formulated according tothe present invention follow.

(i) Antigen Selection and Preparation

Preferably, the antigen to which the antibody binds is a biologicallyimportant glycoprotein and administration of the antibody to a mammalsuffering from a disease or disorder can result in a therapeutic benefitin that mammal. However, antibodies directed against nonpolypeptideantigens (such as tumor-associated glycolipid antigens; see U.S. Pat.No. 5,091,178) are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-b; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-b1, TGF-b2, TGF-b3, TGF-b4, orTGF-b5; a tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins; CD proteinssuch as CD3, CD4, CD8, CD19, CD20, CD22 and CD40; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-alpha, -beta, and -gamma; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 andIL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2,HER3 or HER4 receptor; and fragments of any of the above-listedpolypeptides.

Exemplary molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22,CD34 and CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; B cell surface antigens, such asCD20 or BR3; a member of the tumor necrosis receptor superfamily,including DR5; prostate stem cell antigen (PSCA); cell adhesionmolecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM,alpha4/beta7 integrin, and alphav/beta3 integrin including either alphaor beta subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF as well as receptors therefor;tissue factor (TF); a tumor necrosis factor (TNF) such as TNF-alpha orTNF-beta, alpha interferon (alpha-IFN); an interleukin, such as IL-8;IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor;mpl receptor; CTLA-4; protein C etc.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

For production of HER2 antibodies, the HER2 antigen to be used forproduction thereof may be, e.g., a soluble form of the extracellulardomain of HER2 or a portion thereof, containing the desired epitope.Alternatively, cells expressing HER2 at their cell surface (e.g. NIH-3T3cells transformed to overexpress HER2; or a carcinoma cell line such asSK-BR-3 cells, see Stancovski et al. PNAS (USA) 88:8691-8695 (1991)) canbe used to generate antibodies.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical and/or bind the same epitope, except forpossible variants that may arise during production of the monoclonalantibody. Thus, the modifier “monoclonal” indicates the character of theantibody as not being a mixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Phickthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

WO 01/00245 describes production of exemplary humanized HER2 antibodieswhich bind HER2 and block ligand activation of a HER receptor. Thehumanized antibody of particular interest herein blocks EGF, TGF-αand/or HRG mediated activation of MAPK essentially as effectively asmurine monoclonal antibody 2C4 (or a Fab fragment thereof) and/or bindsHER2 essentially as effectively as murine monoclonal antibody 2C4 (or aFab fragment thereof). The humanized antibody herein may, for example,comprise nonhuman hypervariable region residues incorporated into ahuman variable heavy domain and may further comprise a framework region(FR) substitution at a position selected from the group consisting of69H, 71H and 73H utilizing the variable domain numbering system setforth in Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991). In one embodiment, the humanized antibody comprises FRsubstitutions at two or all of positions 69H, 71H and 73H.

An exemplary humanized antibody of interest herein comprises variableheavy domain complementarity determining residues GFTFTDYTMX, where X ispreferably D or S (SEQ ID No. 7); DVNPNSGGSIYNQRFKG (SEQ ID No. 8);and/or NLGPSFYFDY (SEQ ID No. 9), optionally comprising amino acidmodifications of those CDR residues, e.g. where the modificationsessentially maintain or improve affinity of the antibody. For example,the antibody variant of interest may have from about one to about sevenor about five amino acid substitutions in the above variable heavy CDRsequences. Such antibody variants may be prepared by affinitymaturation, e.g., as described below. The most preferred humanizedantibody comprises the variable heavy domain amino acid sequence in SEQID No. 4.

The humanized antibody may comprise variable light domaincomplementarity determining residues KASQDVSIGVA (SEQ ID No. 10);SASYXXX, where the X as position 5 is preferably R or L, wherein the Xat position 6 is preferably Y or E, and the X as position 7 ispreferably T or S (SEQ ID No. 11); and/or QQYYIYPYT (SEQ ID No. 12),e.g. in addition to those variable heavy domain CDR residues in thepreceding paragraph. Such humanized antibodies optionally comprise aminoacid modifications of the above CDR residues, e.g. where themodifications essentially maintain or improve affinity of the antibody.For example, the antibody variant of interest may have from about one toabout seven or about five amino acid substitutions in the above variablelight CDR sequences. Such antibody variants may be prepared by affinitymaturation, e.g., as described below. The most preferred humanizedantibody comprises the variable light domain amino acid sequence in SEQID No. 3.

The present application also contemplates affinity matured antibodieswhich bind HER2 and block ligand activation of a HER receptor. Theparent antibody may be a human antibody or a humanized antibody, e.g.,one comprising the variable light and/or heavy sequences of SEQ ID Nos.3 and 4, respectively (i.e. variant 574). The affinity matured antibodypreferably binds to HER2 receptor with an affinity superior to that ofmurine 2C4 or variant 574 (e.g. from about two or about four fold, toabout 100 fold or about 1000 fold improved affinity, e.g. as assessedusing a HER2-extracellular domain (ECD) ELISA). Exemplary variable heavyCDR residues for substitution include H28, H30, H34, H35, H64, H96, H99,or combinations of two or more (e.g. two, three, four, five, six, orseven of these residues). Examples of variable light CDR residues foralteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 orcombinations of two or more (e.g. two to three, four, five or up toabout ten of these residues).

Various forms of the humanized antibody or affinity matured antibody arecontemplated. For example, the humanized antibody or affinity maturedantibody may be an antibody fragment, such as a Fab, which is optionallyconjugated with one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody or affinitymatured antibody may be an full length antibody, such as an full lengthIgG1 antibody.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human HER2 antibodies are described in U.S. Pat. No. 5,772,997 issuedJun. 30, 1998 and WO 97/00271 published Jan. 3, 1997.

(v) Antibody fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of full length antibodies (see, e.g., Morimoto et al., Journalof Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The antibody fragmentmay also be a “linear antibody”, e.g., as described in U.S. Pat. No.5,641,870 for example. Such linear antibody fragments may bemonospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the HER2 protein. Other suchantibodies may combine a HER2 binding site with binding site(s) forEGFR, HER3 and/or HER4. Alternatively, a HER2 arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aT-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the HER2-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express HER2. These antibodies possess a HER2-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific HER2/FcγRIII antibody and U.S. Pat.No. 5,837,234 discloses a bispecific HER2/FcγRI antibody IDM1 (Osidem).A bispecific HER2/Fca antibody is shown in WO98/02463. U.S. Pat. No.5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is abispecific HER2-FcγRIII Ab.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein full lengthantibodies are proteolytically cleaved to generate F(ab′)₂ fragments.These fragments are reduced in the presence of the dithiol complexingagent sodium arsenite to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.,148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (V_(H)) connected to alight-chain variable domain (V_(L)) by a linker which is too short toallow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the Antibody are prepared by introducingappropriate nucleotide changes into the Antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the Antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe Antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theAntibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed Antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includea Antibody with an N-terminal methionyl residue or the antibody fused toa cytotoxic polypeptide. Other insertional variants of the Antibodymolecule include the fusion to the N- or C-terminus of the Antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the Antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FR orFc region alterations are also contemplated. Conservative substitutionsare shown in Table 1 under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

-   -   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe        (F), Trp (W), Met (M)    -   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr        (Y), Asn (N), Gln (Q)    -   (3) acidic: Asp (D), Glu (E)    -   (4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the Antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and its antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 A1, Presta, L.See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrateattached to an Fc region of the antibody are referenced in WO03/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO97/30087, Patel et al.See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) concerningantibodies with altered carbohydrate attached to the Fc region thereof.Antibody compositions comprising main species antibody with suchcarbohydrate structures attached to the Fc region are contemplatedherein.

Nucleic acid molecules encoding amino acid sequence variants of theAntibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

(viii) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

To identify an antibody which blocks ligand activation of a HERreceptor, the ability of the antibody to block HER ligand binding tocells expressing the HER receptor (e.g. in conjugation with another HERreceptor with which the HER receptor of interest forms a HERhetero-oligomer) may be determined. For example, cells naturallyexpressing, or transfected to express, HER receptors of the HERhetero-oligomer may be incubated with the antibody and then exposed tolabeled HER ligand. The ability of the HER2 antibody to block ligandbinding to the HER receptor in the HER hetero-oligomer may then beevaluated.

For example, inhibition of HRG binding to MCF7 breast tumor cell linesby HER2 antibodies may be performed using monolayer MCF7 cultures on icein a 24-well-plate format essentially as described in WO01/00245. HER2monoclonal antibodies may be added to each well and incubated for 30minutes. ¹²⁵I-labeled rHRGβ1₁₇₇₋₂₂₄ (25 pm) may then be added, and theincubation may be continued for 4 to 16 hours. Dose response curves maybe prepared and an IC₅₀ value may be calculated for the antibody ofinterest. In one embodiment, the antibody which blocks ligand activationof an HER receptor will have an IC50 for inhibiting HRG binding to MCF7cells in this assay of about 50 nM or less, more preferably 10 nM orless. Where the antibody is an antibody fragment such as a Fab fragment,the IC50 for inhibiting HRG binding to MCF7 cells in this assay may, forexample, be about 100 nM or less, more preferably 50 nM or less.

Alternatively, or additionally, the ability of the HER2 antibody toblock HER ligand-stimulated tyrosine phosphorylation of a HER receptorpresent in a HER hetero-oligomer may be assessed. For example, cellsendogenously expressing the HER receptors or transfected to expressedthem may be incubated with the antibody and then assayed for HERligand-dependent tyrosine phosphorylation activity using ananti-phosphotyrosine monoclonal (which is optionally conjugated with adetectable label). The kinase receptor activation assay described inU.S. Pat. No. 5,766,863 is also available for determining HER receptoractivation and blocking of that activity by an antibody.

In one embodiment, one may screen for an antibody which inhibits HRGstimulation of p180 tyrosine phosphorylation in MCF7 cells essentiallyas described in WO01/00245. For example, the MCF7 cells may be plated in24-well plates and monoclonal antibodies to HER2 may be added to eachwell and incubated for 30 minutes at room temperature; thenrHRGβ1₁₇₇₋₂₄₄ may be added to each well to a final concentration of 0.2nM, and the incubation may be continued for 8 minutes. Media may beaspirated from each well, and reactions may be stopped by the additionof 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl,pH 6.8). Each sample (25 μl) may be electrophoresed on a 4-12% gradientgel (Novex) and then electrophoretically transferred to polyvinylidenedifluoride membrane. Antiphosphotyrosine (at 1 pg/ml) immunoblots may bedeveloped, and the intensity of the predominant reactive band atM_(r)˜180,000 may be quantified by reflectance densitometry. Theantibody selected will preferably significantly inhibit HRG stimulationof p180 tyrosine phosphorylation to about 0-35% of control in thisassay. A dose-response curve for inhibition of HRG stimulation of p180tyrosine phosphorylation as determined by reflectance densitometry maybe prepared and an IC₅₀ for the antibody of interest may be calculated.In one embodiment, the antibody which blocks ligand activation of a HERreceptor will have an IC₅₀ for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay of about 50 nM or less, morepreferably 10 nM or less. Where the antibody is an antibody fragmentsuch as a Fab fragment, the IC50 for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay may, for example, be about 100 nMor less, more preferably 50 nM or less.

One may also assess the growth inhibitory effects of the antibody onMDA-MB-175 cells, e.g, essentially as described in Schaefer et al.Oncogene 15:1385-1394 (1997). According to this assay, MDA-MB-175 cellsmay treated with a HER2 monoclonal antibody (10 pg/mL) for 4 days andstained with crystal violet. Incubation with a HER2 antibody may show agrowth inhibitory effect on this cell line similar to that displayed bymonoclonal antibody 2C4. In a further embodiment, exogenous HRG will notsignificantly reverse this inhibition. Preferably, the antibody will beable to inhibit cell proliferation of MDA-MB-175 cells to a greaterextent than monoclonal antibody 4D5 (and optionally to a greater extentthan monoclonal antibody 7F3), both in the presence and absence ofexogenous HRG.

In one embodiment, the HER2 antibody of interest may block heregulindependent association of HER2 with HER3 in both MCF7 and SK-BR-3 cellsas determined in a co-immunoprecipitation experiment such as thatdescribed in WO01/00245 substantially more effectively than monoclonalantibody 4D5, and preferably substantially more effectively thanmonoclonal antibody 7F3.

To identify growth inhibitory HER2 antibodies, one may screen forantibodies which inhibit the growth of cancer cells which overexpressHER2. In one embodiment, the growth inhibitory antibody of choice isable to inhibit growth of SK-BR-3 cells in cell culture by about 20-100%and preferably by about 50-100% at an antibody concentration of about0.5 to 30 pg/ml. To identify such antibodies, the SK-BR-3 assaydescribed in U.S. Pat. No. 5,677,171 can be performed. According to thisassay, SK-BR-3 cells are grown in a 1:1 mixture of F12 and DMEM mediumsupplemented with 10% fetal bovine serum, glutamine and penicillinstreptomycin. The SK-BR-3 cells are plated at 20,000 cells in a 35 mmcell culture dish (2 mls/35 mm dish). 0.5 to 30 pg/ml of the HER2antibody is added per dish. After six days, the number of cells,compared to untreated cells are counted using an electronic COULTER™cell counter. Those antibodies which inhibit growth of the SK-BR-3 cellsby about 20-100% or about 50-100% may be selected as growth inhibitoryantibodies. See U.S. Pat. No. 5,677,171 for assays for screening forgrowth inhibitory antibodies, such as 4D5 and 3E8.

In order to select for HER2 antibodies which induce apoptosis, anannexin binding assay using BT474 cells is available. The BT474 cellsare cultured and seeded in dishes as discussed in the precedingparagraph. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the monoclonal antibody.Following a three day incubation period, monolayers are washed with PBSand detached by trypsinization. Cells are then centrifuged, resuspendedin Ca²⁺ binding buffer and aliquoted into tubes as discussed above forthe cell death assay. Tubes then receive labeled annexin (e.g. annexinV-FTIC) (1 μg/ml). Samples may be analyzed using a FACSCAN™ flowcytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Thoseantibodies which induce statistically significant levels of annexinbinding relative to control are selected as apoptosis-inducingantibodies. In addition to the annexin binding assay, a DNA stainingassay using BT474 cells is available. In order to perform this assay,BT474 cells which have been treated with the antibody of interest asdescribed in the preceding two paragraphs are incubated with 9 μg/mlHOECHST 33342™ for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flowcytometer (Coulter Corporation) using MODFIT LT™ software (VeritySoftware House). Antibodies which induce a change in the percentage ofapoptotic cells which is 2 fold or greater (and preferably 3 fold orgreater) than untreated cells (up to 100% apoptotic cells) may beselected as pro-apoptotic antibodies using this assay. See WO98/17797for assays for screening for HER2 antibodies which induce apoptosis,such as 7C2 and 7F3.

To screen for antibodies which bind to an epitope on HER2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed to assesswhether the antibody cross-blocks binding of an antibody, such as 2C4 orPertuzumab, to HER2. Alternatively, or additionally, epitope mapping canbe performed by methods known in the art and/or one can study theantibody-HER2 structure (Franklin et al. Cancer Cell 5:317-328 (2004))to see what domain(s) of HER2 is/are bound by the antibody.

(ix) Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. a small molecule toxin or an enzymatically active toxin ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin, amaytansine (U.S. Pat. No. 5,208,020), a trichothene, and CC1065 are alsocontemplated herein.

In one preferred embodiment of the invention, the antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) togenerate a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises a HER2 antibody conjugatedto one or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ₁ ¹, α₂ ¹, α₃ ¹,N-acetyl-γ₁ ¹, PSAG and θ¹ ₁ (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)).See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001expressly incorporated herein by reference.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated HER2 antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the HER2 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

(x) Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta, L.) describes antibodies with improved ADCC functionin the presence of human effector cells, where the antibodies compriseamino acid substitutions in the Fc region thereof. Preferably, theantibody with improved ADCC comprises substitutions at positions 298,333, and/or 334 of the Fc region. Preferably the altered Fc region is ahuman IgG1 Fc region comprising or consisting of substitutions at one,two or three of these positions.

Antibodies with altered C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. Antibodies with substitutions in an Fc region thereofand increased serum half-lives are also described in WO00/42072 (Presta,L.).

Engineered antibodies with three or more (preferably four) functionalantigen binding sites are also contemplated (US Appln No. US2002/0004587A1, Miller et al.).

The HER2 antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; andWO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine PEG-PE). Liposomes are extruded through filtersof defined pore size to yield liposomes with the desired diameter. Fab′fragments of the antibody of the present invention can be conjugated tothe liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288(1982) via a disulfide interchange reaction. A chemotherapeutic agent isoptionally contained within the liposome. See Gabizon et al. J. NationalCancer Inst. 81(19) 1484 (1989).

(ix) Exemplary Antibodies

Exemplary antibodies which can be formulated according to the presentinvention include, but are not limited to the following:

anti-ErbB antibodies, including anti-HER2 antibodies, such as thosedescribed in more detail herein; antibodies that bind to a B-cellsurface marker, such as CD19, CD20 (for example Rituximab (RITUXAN®) andhumanized 2H7), CD22, CD40 or BR3;antibodies that bind to IgE, including Omalizumab (XOLAIR®) commerciallyavailable from Genentech, E26 (FIGS. 17A-B herein), HAE1 (FIGS. 17A-Bherein), IgE antibody with an amino acid substitution at position 265 ofan Fc region thereof (US 2004/0191244 A1), Hu-901 (FIGS. 17A-B herein),an IgE antibody as in WO2004/070011, or an antibody (including antibodyfragments and full length antibodies) comprising the variable domains ofany of those IgE antibodies. See, also, Presta et al., J. Immunol.151:2623-2632 (1993); International Publication No. WO 95/19181; U.S.Pat. No. 5,714,338, issued Feb. 3, 1998; U.S. Pat. No. 5,091,313, issuedFeb. 25, 1992; WO 93/04173 published Mar. 4, 1993; WO 99/01556 publishedJan. 14, 1999; and U.S. Pat. No. 5,714,338;antibodies that bind to vascular endothelial growth factor (VEGF) or areceptor thereof, including Bevacizumab (AVASTIN™), commerciallyavailable from Genentech, and Ranibizumab (LUCENTIS™);anti-IL-8 antibodies (St John et al., Chest, 103:932 (1993), andInternational Publication No. WO 95/23865); anti-PSCA antibodies(WO01/40309);anti-CD40 antibodies, including S2C6 and humanized variants thereof(WO00/75348);anti-CD11a antibodies, including efalizumab (RAPTIVA®) (U.S. Pat. No.5,622,700, WO 98/23761, Steppe et al., Transplant Intl. 4:3-7 (1991),and Hourmant et al., Transplantation 58:377-380 (1994)); anti-CD18antibodies (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, or as in WO97/26912, published Jul. 31, 1997);anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998);anti-TNF-alpha antibodies including cA2 (REMICADE®), CDP571 and MAK-195(See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997, Lorenz et al. J.Immunol. 156(4):1646-1653 (1996), and Dhainaut et al. Crit. Care Med.23(9):1461-1469 (1995));anti-Tissue Factor (TF) (European Patent No. 0 420 937 B1 granted Nov.9, 1994);anti-human α₄β₇ integrin (WO 98/06248 published Feb. 19, 1998);anti-EGFR antibodies, including chimerized or humanized 225 antibody asin WO 96/40210 published Dec. 19, 1996;anti-CD3 antibodies, such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7,1985);anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and(ZENAPAX®) (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997);anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. ArthritisRheum 39(1):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H(Riechmann et al. Nature 332:323-337 (1988);anti-Fc receptor antibodies such as the M22 antibody directed againstFcγRI as in Graziano et al. J. Immunol. 155(10):4996-5002 (1995);anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkeyet al. Cancer Res. 55(23Suppl): 5935s-5945s (1995);antibodies directed against breast epithelial cells including huBrE-3,hu-Mc 3 and CHL6 (Ceriani et al. Cancer Res. 55(23): 5852s-5856s (1995);and Richman et al. Cancer Res. 55(23 Supp): 5916s-5920s (1995));antibodies that bind to colon carcinoma cells such as C242 (Litton etal. Eur J. Immunol. 26(1):1-9 (1996));anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al. J. Immunol.155(2):925-937 (1995));anti-CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55(23Suppl):5908s-5910s (1995) and CMA-676 or CDP771;anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al. Cancer Res55(23 Suppl):5899s-5907s (1995);anti-EpCAM antibodies such as 17-1A (PANOREX®);anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO®);anti-RSV antibodies such as MEDI-493 (SYNAGIS®);anti-CMV antibodies such as PROTOVIR®;anti-HIV antibodies such as PRO542;anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR®;anti-CA 125 antibody OvaRex;anti-idiotypic GD3 epitope antibody BEC2;anti-αvβ3 antibody VITAXIN®;anti-human renal cell carcinoma antibody such as ch-G250; ING-1;anti-human 17-1A antibody (3622W94);anti-human colorectal tumor antibody (A33);anti-human melanoma antibody R24 directed against GD3 ganglioside;anti-human squamous-cell carcinoma (SF-25); andanti-human leukocyte antigen (HLA) antibodies such as Smart ID10 and theanti-HLA DR antibody Oncolym (Lym-1).

(xi) Antibody Variant Compositions

The present invention, in at least one aspect, concerns formulationscomprising a composition which comprises a mixture of a main speciesantibody and one or more variants thereof. Where the main speciesantibody binds HER2, preferably the HER2 antibody (either or both of themain species HER2 antibody and antibody variant thereof) is one whichbinds to Domain II of HER2, inhibits HER dimerization more effectivelythan Trastuzumab, and/or binds to a heterodimeric binding site of HER2.The preferred embodiment herein of the main species antibody is onecomprising the variable light and variable heavy amino acid sequences inSEQ ID Nos. 3 and 4, and most preferably comprising a light chain aminoacid sequence selected from SEQ ID No. 15 and 23, and a heavy chainamino acid sequence selected from SEQ ID No. 16 and 24.

In one embodiment, the formulated HER2 antibody composition comprises amixture of the main species HER2 antibody and an amino acid sequencevariant thereof comprising an amino-terminal leader extension.Preferably, the amino-terminal leader extension is on a light chain ofthe antibody variant (e.g. on one or two light chains of the antibodyvariant). The main species HER2 antibody or the antibody variant may bean full length antibody or antibody fragment (e.g. Fab of F(ab′)₂fragments), but preferably both are full length antibodies. The antibodyvariant herein may comprise an amino-terminal leader extension on anyone or more of the heavy or light chains thereof. Preferably, theamino-terminal leader extension is on one or two light chains of theantibody. The amino-terminal leader extension preferably comprises orconsists of VHS-. Presence of the amino-terminal leader extension in thecomposition can be detected by various analytical techniques including,but not limited to, N-terminal sequence analysis, assay for chargeheterogeneity (for instance, cation exchange chromatography or capillaryzone electrophoresis), mass spectrometry, etc. The amount of theantibody variant in the composition generally ranges from an amount thatconstitutes the detection limit of any assay (preferably N-terminalsequence analysis) used to detect the variant to an amount less than theamount of the main species antibody. Generally, about 20% or less (e.g.from about 1% to about 15%, for instance from 5% to about 15%) of theantibody molecules in the composition comprise an amino-terminal leaderextension. Such percentage amounts are preferably determined usingquantitative N-terminal sequence analysis or cation exchange analysis(preferably using a high-resolution, weak cation-exchange column, suchas a PROPAC WCX-10™ cation exchange column). Aside from theamino-terminal leader extension variant, further amino acid sequencealterations of the main species antibody and/or variant arecontemplated, including but not limited to an antibody comprising aC-terminal lysine residue on one or both heavy chains thereof, adeamidated antibody variant, etc.

Moreover, the main species antibody or variant may further compriseglycosylation variations, non-limiting examples of which include HER2antibody comprising a G1 or G2 oligosaccharide structure attached to theFc region thereof, HER2 antibody comprising a carbohydrate moietyattached to a light chain thereof (e.g. one or two carbohydrate moietiesattached to one or two light chains of the antibody), HER2 antibodycomprising a non-glycosylated heavy chain.

III. Preparation of the Formulation

The present invention provides, in a first aspect, a stablepharmaceutical formulation comprising a monoclonal antibody, preferablya full length human or humanized IgG1 antibody, in histidine-acetatebuffer, pH 5.5 to 6.5, preferably pH 5.8 to 6.2. However, the antibodyin the formulation may be an antibody fragment comprising anantigen-binding region, such as a Fab or F(ab′)₂ fragment.

In another embodiment, the invention concerns a pharmaceuticalformulation comprising, or consisting essentially of, a full length IgG1antibody susceptible to deamidation or aggregation in an amount fromabout 10 mg/mL to about 250 mg/mL; histidine-acetate buffer, pH 5.5 to6.5; saccharide selected from the group consisting of trehalose andsucrose, in an amount from about 60 mM to about 250 mM; and polysorbate20 in an amount from about 0.01% to about 0.1%.

In yet a further embodiment, the invention provides a pharmaceuticalformulation comprising an antibody that binds to domain II of HER2 in ahistidine buffer at a pH from about 5.5 to about 6.5, a saccharide and asurfactant. For example, the formulation may comprise Pertuzumab in anamount from about 20 mg/mL to about 40 mg/mL, histidine-acetate buffer,sucrose, and polysorbate 20, wherein the pH of the formulation is fromabout 5.5 to about 6.5

In another aspect, the invention provides a pharmaceutical formulationcomprising a DR5 antibody in a histidine buffer at a pH from about 5.5to about 6.5, a saccharide, and a surfactant. Such a formulation may,for example, comprise, Apomab in an amount from about 10 mg/mL to about30 mg/mL, histidine-acetate buffer, trehalose, and polysorbate 20,wherein the pH of the formulation is from about 5.5 to about 6.5.

The formulation is especially useful for antibodies that are susceptibleto deamidation and/or aggregation and/or fragmentation, in that thebuffer retards deamidation and/or aggregation and/or fragmentation ofthe antibody formulated therein. In addition, unlike other histidinebuffers prepared using HCl, the histidine-acetate buffer lacks thechloride ion which was found to be beneficial herein in that this bufferwhen combined with saccharide had the same protective effect on antibodyas polysorbate 20, and was stable and compatible with storage instainless steel tanks. Thus, in addition to the formulation per secomprising the antibody susceptible to deamidation, aggregation and/orfragmentation, the invention provides a method for reducing deamidation,aggregation and/or fragmentation of a therapeutic monoclonal antibody(for example, relative to a composition at a different pH or in adifferent buffer), comprising formulating the antibody in ahistidine-acetate buffer, pH 5.5 to 6.5. In this embodiment, one maydetermine or measure deamidation, aggregation and/or fragmentationbefore and after the antibody is formulated, with the formulatedantibody demonstrating acceptable deamidation, aggregation and/orfragmentation in the formulation and upon storage thereof.

The antibody in the formulation may bind an antigen including but notlimited to: HER2, CD20, IgE, DR5, BR3 and VEGF.

Where the formulated antibody binds HER2, it preferably is one whichbinds to Domain II of HER2, inhibits HER dimerization more effectivelythan Trastuzumab, and/or binds to a heterodimeric binding site of HER2.The preferred embodiment herein of a formulated HER2 antibody is onecomprising the variable light and variable heavy amino acid sequences inSEQ ID Nos. 3 and 4, and most preferably comprising the light chain andheavy chain amino acid sequences in SEQ ID Nos. 15 and 16 (Pertuzumab).

Examples of CD20 antibodies which can be formulated herein include:“C2B8” which is now called “Rituximab” (“RITUXAN®”) commerciallyavailable from Genentech (see also U.S. Pat. No. 5,736,137, expresslyincorporated herein by reference); the yttrium-[90]-labeled 2B8 murineantibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN®commercially available from Biogen-Idec (see also U.S. Pat. No.5,736,137, expressly incorporated herein by reference); murine IgG2a“B1,” also called “Tositumomab,” optionally labeled with ¹³¹I togenerate the “131I-B1” antibody (iodine 1131 tositumomab, BEXXAR™) (U.S.Pat. No. 5,595,721, expressly incorporated herein by reference); murinemonoclonal antibody “1F5” (Press et al. Blood 69(2):584-591 (1987) andvariants thereof including “framework patched” or humanized 1F5(WO03/002607, Leung, S.); ATCC deposit HB-96450); murine 2H7 andchimeric 2H7 antibody (Clark et al. PNAS 82: 1766-1770 (1985); U.S. Pat.No. 5,500,362, expressly incorporated herein by reference); humanized2H7; huMax-CD20 (WO 04/035607, Genmab, Denmark); AME-133 (AppliedMolecular Evolution); A20 antibody or variants thereof such as chimericor humanized A20 antibody (cA20, hA20, respectively) (US 2003/0219433,Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 orNU-B2 available from the International Leukocyte Typing Workshop(Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440,Oxford University Press (1987)).

In the preferred embodiment of a formulated CD20 antibody, the CD20antibody is a humanized 2H7 antibody. Preferred humanized 2H7 antibodiesherein are 2H7v16 and 2H7v511. The humanized 2H7v16 may be an intactantibody or antibody fragment comprising the variable light and variableheavy sequences in FIGS. 18A-B (SEQ ID Nos. 26 and 29). Where thehumanized 2H7v16 antibody is a full length antibody, preferably itcomprises the light and heavy chain amino acid sequences with SEQ IDNos. 63 and 65.

Where the antibody binds VEGF, it preferably comprises the variabledomain sequences as depicted in FIG. 19 . The most preferred anti-VEGFantibody is full length humanized IgG1 antibody, Bevacizumab (AVASTIN™)commercially available from Genentech.

Where the formulated antibody binds IgE, it is preferably selected fromthe group consisting of: E25, Omalizumab (XOLAIR®) commerciallyavailable from Genentech (see also FIGS. 17A-B), E26 (FIGS. 17A-Bherein), HAE1 (FIGS. 17A-B herein), IgE antibody with an amino acidsubstitution at position 265 of an Fc region thereof (US 2004/0191244A1), Hu-901 (FIGS. 17A-B herein), an IgE antibody as in WO2004/070011,or an antibody (including antibody fragments and full length antibodies)comprising the variable domains of any of those IgE antibodies.

Where the antibody binds to a receptor in the tumor necrosis factor(TNF) superfamily or to a death receptor, it preferably binds to DR5,and preferably is an agonist antibody. Publications in this area includeSheridan et al., Science, 277:818-821 (1997), Pan et al., Science,277:815-818 (1997), WO98/51793 published Nov. 19, 1998; WO98/41629published Sep. 24, 1998; Screaton et al., Curr. Biol., 7:693-696 (1997);Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., NatureGenetics, 17:141-143 (1997); WO98/35986 published Aug. 20, 1998;EP870,827 published Oct. 14, 1998; WO98/46643 published Oct. 22, 1998;WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25, 1999;WO99/11791 published Mar. 11, 1999; US 2002/0072091 published Aug. 13,2002; US 2002/0098550 published Dec. 7, 2001; U.S. Pat. No. 6,313,269issued Dec. 6, 2001; US 2001/0010924 published Aug. 2, 2001; US2003/01255540 published Jul. 3, 2003; US 2002/0160446 published Oct. 31,2002, US 2002/0048785 published Apr. 25, 2002; U.S. Pat. No. 6,342,369issued February, 2002; U.S. Pat. No. 6,569,642 issued May 27, 2003, U.S.Pat. No. 6,072,047 issued Jun. 6, 2000, U.S. Pat. No. 6,642,358 issuedNov. 4, 2003; U.S. Pat. No. 6,743,625 issued Jun. 1, 2004. The mostpreferred DR5 antibody is Apomab.

Each of the formulations noted above comprises a buffer, preferably ahistidine buffer, and most preferably a histidine-acetate buffer with apH of 5.5 to 6.5, preferably 5.8 to 6.2, for example approximately 6.0.The concentration of the buffer is dictated, at least in part, by thedesired pH. Exemplary concentrations for the buffer are in the rangefrom about 1 mM to about 200 mM, preferably from about 10 mM to about 40mM, most preferably about 20 mM.

The antibody concentration in the formulation is preferably in the rangefrom about 10 mg/mL to about 250 mg/mL. The antibody concentration maybe determined based on the intended use and mode of administration ofthe formulation. For example, where the formulation is for IVadministration (e.g. a HER2 antibody), the antibody concentration in theformulation is preferably from about 20 mg/mL to about 40 mg/mL. In theexemplified Pertuzumab formulation intended for intravenous (IV)administration, the antibody concentration was from about 20 mg/mL toabout 40 mg/mL, most preferably about 30 mg/mL.

Where the antibody is for SQ or IM administration (e.g. for an anti-IgEantibody) higher concentrations of the antibody may be desired. Suchsubstantially high antibody concentrations may be from about 50 mg/mL toabout 250 mg/mL, or from about 80 mg/mL to about 250 mg/mL, or fromabout 100 mg/mL to about 200 mg/mL.

Where the formulation comprises a DR5 antibody, such as Apomab,exemplary antibody concentrations are from about 10 mg/mL to about 30mg/mL, for example about 20 mg/mL DR5 antibody; such formulation beinguseful for intravenous administration.

The formulation for administration is preferably an aqueous formulation(not lyophilized) and has not been subjected to prior lyophilization.While the formulation may be lyophilized, preferably it is not. However,freezing of the aqueous formulation, without simultaneous drying thatoccurs during freeze-drying, is specifically contemplated herein,facilitating longer term storage thereof, for instance in a stainlesssteel tank.

The formulation preferably further comprises a saccharide, mostpreferably a disaccharide, such as trehalose or sucrose. The saccharideis generally included in an amount which reduces soluble aggregateformation, such as that which occurs upon freeze/thaw. Exemplarysaccharide concentrations are in the range from about 10 mM to about 1M,for example from about 60 mM to about 250 mM, and most preferably about120 mM for a HER2 antibody formulation, and about 240 mM for a DR5antibody formulation.

While it was found herein that a formulation comprisinghistidine-acetate buffer and saccharide was stable, the formulationoptionally further comprises surfactant, such as polysorbate, mostpreferably polysorbate 20. The surfactant is generally included in anamount which reduces insoluble aggregate formation (such as that whichoccurs upon shaking or shipping). The surfactant concentration ispreferably from about 0.0001% to about 1.0%, most preferably from about0.01% to about 0.1%, for example about 0.02%.

Optionally, the formulation does not contain a tonicifying amount of asalt such as sodium chloride.

The formulation is generally sterile, and this can be achieved accordingto the procedures known to the skilled person for generating sterilepharmaceutical formulations suitable for administration to humansubjects, including filtration through sterile filtration membranes,prior to, or following, preparation of the formulation.

Moreover, the formulation is desirably one which has been demonstratedto be stable upon storage. Various stability assays are available to theskilled practitioner for confirming the stability of the formulation.For example, the formulation may be one which is found to be stable uponstorage: at about 40° C. for at least 4 weeks; at about 5° C. or about15° C. for at least 3 months or at least 1 year; and/or about −20° C.for at least 3 months. Stability can be tested by evaluating physicalstability, chemical stability, and/or biological activity of theantibody in the formulation around the time of formulation as well asfollowing storage at the noted temperatures. Physical and/or stabilitycan be evaluated qualitatively and/or quantitatively in a variety ofdifferent ways, including evaluation of aggregate formation (for exampleusing size exclusion chromatography, by measuring turbidity, and/or byvisual inspection); by assessing charge heterogeneity using cationexchange chromatography or capillary zone electrophoresis;amino-terminal or carboxy-terminal sequence analysis; mass spectrometricanalysis; SDS-PAGE analysis to compare reduced and intact antibody;peptide map (for example tryptic or LYS-C) analysis; evalutingbiological activity or antigen binding function of the antibody; etc.Instability may result in aggregation, deamidation (e.g. Asndeamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Aspisomeriation), clipping/hydrolysis/fragmentation (e.g. hinge regionfragmentation), succinimide formation, unpaired cysteine(s), N-terminalextension, C-terminal processing, glycosylation differences, etc.Biological activity or antigen binding function can be evaluated usingvarious techniques available to the skilled practitioner.

As noted above, freezing of the formulation is specifically contemplatedherein. Hence, the formulation can be tested for stability upon freezingand thawing.

According, the invention also provides a method of making apharmaceutical formulation comprising preparing the formulation asdescribed herein, and evaluating physical stability, chemical stability,or biological activity of the monoclonal antibody in the formulation.

In the preferred embodiment, the formulation is provided inside a vialwith a stopper pierceable by a syringe, preferably in aqueous form. Thevial is desirably stored at about 2-8° C. until it is administered to asubject in need thereof. The vial may for example be a 20 cc vial (forexample for a 420 mg dose) or 50 cc vial (for example for a 1050 mgdose). For a DR5 antibody, such as Apomab, the formulation may beprovided in a 5 cc glass vial (e.g. 5.5 ml fill).

In another embodiment, the formulation is provided inside a stainlesssteel tank. The formulation in the stainless steel tank is optionallyfrozen and not freeze-dried.

One or more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; co-solvents;antioxidants including ascorbic acid and methionine; chelating agentssuch as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradablepolymers such as polyesters; preservatives; and/or salt-formingcounterions such as sodium.

IV. Treatment with the Antibody Formulation

In one embodiment, the invention provides a method of treating a diseaseor disorder in a subject comprising administering the formulationdescribed herein to a subject in an amount effective to treat thedisease or disorder.

Where the antibody in the formulation binds to HER2, it is preferablyused to treat cancer. The cancer will generally comprise HER2-expressingcells, such that the HER2 antibody herein is able to bind to the cancercells. Thus, the invention in this embodiment concerns a method fortreating HER2-expressing cancer in a subject, comprising administeringthe HER2 antibody pharmaceutical formulation to the subject in an amounteffective to treat the cancer. Various cancers that can be treated withthe composition are listed in the definitions section above.

It is also contemplated that the HER2 antibody formulation may be usedto treat various non-malignant diseases or disorders, such a includeautoimmune disease (e.g. psoriasis); endometriosis; scleroderma;restenosis; polyps such as colon polyps, nasal polyps orgastrointestinal polyps; fibroadenoma; respiratory disease (seedefinition above); cholecystitis; neurofibromatosis; polycystic kidneydisease; inflammatory diseases; skin disorders including psoriasis anddermatitis; vascular disease (see definition above); conditionsinvolving abnormal proliferation of vascular epithelial cells;gastrointestinal ulcers; Menetrier's disease, secreting adenomas orprotein loss syndrome; renal disorders; angiogenic disorders; oculardisease such as age related macular degeneration, presumed ocularhistoplasmosis syndrome, retinal neovascularization from proliferativediabetic retinopathy, retinal vascularization, diabetic retinopathy, orage related macular degeneration; bone associated pathologies such asosteoarthritis, rickets and osteoporosis; damage following a cerebralischemic event; fibrotic or edemia diseases such as hepatic cirrhosis,lung fibrosis, carcoidosis, throiditis, hyperviscosity syndromesystemic, Osler Weber-Rendu disease, chronic occlusive pulmonarydisease, or edema following burns, trauma, radiation, stroke, hypoxia orischemia; hypersensitivity reaction of the skin; diabetic retinopathyand diabetic nephropathy; Guillain-Barre syndrome; graft versus hostdisease or transplant rejection; Paget's disease; bone or jointinflammation; photoaging (e.g. caused by UV radiation of human skin);benign prostatic hypertrophy; certain microbial infections includingmicrobial pathogens selected from adenovirus, hantaviruses, Borreliaburgdorferi, Yersinia spp. and Bordetella pertussis; thrombus caused byplatelet aggregation; reproductive conditions such as endometriosis,ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterinebleeding, or menometrorrhagia; synovitis; atheroma; acute and chronicnephropathies (including proliferative glomerulonephritis anddiabetes-induced renal disease); eczema; hypertrophic scar formation;endotoxic shock and fungal infection; familial adenomatosis polyposis;neurodedenerative diseases (e.g. Alzheimer's disease, AIDS-relateddementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration);myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis ofthe lung, kidney or liver; T-cell mediated hypersensitivity disease;infantile hypertrophic pyloric stenosis; urinary obstructive syndrome;psoriatic arthritis; and Hasimoto's thyroiditis. Preferred non-malignantindications for therapy herein include psoriasis, endometriosis,scleroderma, vascular disease (e.g. restenosis, artherosclerosis,coronary artery disease, or hypertension), colon polyps, fibroadenoma orrespiratory disease (e.g. asthma, chronic bronchitis, bronchieactasis orcystic fibrosis).

Where the antibody in the formulation binds to a B-cell surface markersuch as CD20 or BR3, the formulation may be used to treat a B-cellmalignancy, such as NHL or CLL, an autoimmune disease, graft rejection,or to block an immune response to a foreign antigen, such as anantibody, a toxin, a gene therapy viral vector, a graft, an infectiousagent, or an alloantigen (see WO 01/03734, Grillo-Lopez et al.).

Where the antibody in the formulation is an IgE antibody, it may be usedto treat an IgE-mediated disorder (USSN 2004/0197324 A1, Liu and Shire),such as allergic asthma, allergic rhinitis, atopic dermatitis, allergicgastroenteropathy, hypersensitivity, eczema, urticaria, allergicbronchopulmonary aspergillosis, parasitic disease, hyper-IgE syndrome,ataxia-telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia,IgE myeloma, and graft-versus-host reaction.

Antibodies that bind to a receptor in the TNF superfamily (for instancewhich bind to DR5), or which bind to VEGF (or a receptor thereof), maybe used to treat cancer, various forms of which are described in thedefinitions section above. Preferably, the cancer treated with a DR5antibody formulation is a solid tumor or NHL.

Where the indication is cancer, the patient may be treated with acombination of the antibody formulation, and a chemotherapeutic agent.The combined administration includes coadministration or concurrentadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Thus, thechemotherapeutic agent may be administered prior to, or following,administration of the composition. In this embodiment, the timingbetween at least one administration of the chemotherapeutic agent and atleast one administration of the composition is preferably approximately1 month or less, and most preferably approximately 2 weeks or less.Alternatively, the chemotherapeutic agent and the composition areadministered concurrently to the patient, in a single formulation orseparate formulations.

Treatment with the formulation will result in an improvement in thesigns or symptoms of cancer or disease. For instance, where the diseasebeing treated is cancer, such therapy may result in an improvement insurvival (overall survival and/or progression free survival) and/or mayresult in an objective clinical response (partial or complete).Moreover, treatment with the combination of the chemotherapeutic agentand the antibody formulation may result in a synergistic, or greaterthan additive, therapeutic benefit to the patient.

Preferably, the antibody in the formulation administered is a nakedantibody. However, the antibody administered may be conjugated with acytotoxic agent. Preferably, the immunoconjugate and/or antigen to whichit is bound is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the cancer cellto which it binds. In a preferred embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the cancer cell. Examples ofsuch cytotoxic agents include maytansinoids, calicheamicins,ribonucleases and DNA endonucleases.

The formulation is administered to a human patient in accord with knownmethods, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous, intramuscular or subcutaneous administration of antibodycomposition is preferred, with intravenous administration being mostpreferred.

For subcutaneous delivery, the formulation may be administered viasyringe; injection device (e.g. the INJECT-EASE™ and GENJECT™ device);injector pen (such as the GENPEN™); needleless device (e.g. MEDIJECTOR™and BIOJECTOR™); or subcutaneous patch delivery system.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of HER2 or DR5 antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. The dosage of the antibody will generally be in the range fromabout 0.05 mg/kg to about 10 mg/kg. If a chemotherapeutic agent isadministered, it is usually administered at dosages known therefor, oroptionally lowered due to combined action of the drugs or negative sideeffects attributable to administration of the chemotherapeutic agent.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).

Other therapeutic regimens may be combined with the antibody including,but not limited to: a second (third, fourth, etc) chemotherapeuticagent(s) (i.e. “cocktails” of different chemotherapeutic agents);another monoclonal antibody; a growth inhibitory agent; a cytotoxicagent; a chemotherapeutic agent; EGFR-targeted drug; tyrosine kinaseinhibitor; anti-angiogenic agent; and/or cytokine; etc.

In addition to the above therapeutic regimes, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

V. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the pharmaceutical formulation of the presentinvention and provides instructions for its use. The article ofmanufacture comprises a container. Suitable containers include, forexample, bottles, vials (e.g. dual chamber vials), syringes (such asdual chamber syringes) and test tubes. The container may be formed froma variety of materials such as glass or plastic. The container holds theformulation and the label on, or associated with, the container mayindicate directions for use. The container holding the formulation maybe a multi-use vial, which allows for repeat administrations (e.g. from2-6 administrations) of the reconstituted formulation. The article ofmanufacture may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse as noted in the previous section.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

EXAMPLES Stable Pertuzumab Liquid Formulations

These examples describe the development and stability testing of stableliquid formulations comprising Pertuzumab at protein concentrations inthe range from about 10 mg/mL-180 mg/mL. The selected formulations hadlow turbidity, and were physically and chemically stable. A chloride ionwas removed from the formulation to reduce the risk of corrosion. Theformulation was isotonic, and suitable for subcutaneous or intramusculardelivery. Insoluble aggregate formation upon agitation stress wasprevented using histidine-acetate and sucrose formulation, without theneed to include polysorbate 20.

Analytical Methods Color, Appearance and Clarity (CAC)

The color, appearance, and clarity of the samples were determined byvisual inspection of vials against a white and black background underwhite fluorescence light at room temperature.

UV Concentration Measurements

The liquid product aliquot was first diluted with formulation buffer sothat the A_(max) near 278 nm is within 0.5-1.0 absorbance unit. The UVabsorbance of the diluted samples was measured in a quartz cuvette with1 cm path length on an HP 8453 spectrophotometer. Absorbance wasmeasured at 278 nm and 320 nm. The absorbance from 320 nm is used tocorrect background light scattering due to larger aggregates, bubblesand particles. The measurements were blanked against the formulationbuffer. The protein concentration was determined using the absorptivityof 1.50 (mg/mL)⁻¹ cm⁻¹.

pH Measurements

The pH was measured at room temperature using a RADIOMETER COPENHAGENPHM82™ pH meter. The probe used was a combined glass/reference electrodewith radiometer connector (Sigma, Cat #E-5759). Standard solutions of pH4.01 and pH 7.00 (EM Science) were used for calibration of the pH meter.

Ion-Exchange Chromatography (IEX)

Cation exchange chromatography was employed to measure changes in chargevariants. This assay utilizes a DIONEX PROPAC WCX-1O™ column on an HP1100™ HPLC system. Samples were diluted to 1 mg/mL with the mobile phaseA containing 20 mM MES at pH 6.0. 50 mL of diluted samples were thenloaded on the column that was kept at ambient temperature. The peakswere eluted with a shallow NaCl gradient using mobile B containing 20 mMMES, 250 mM NaCl, pH 6.0. The eluent was monitored at 280 nm. The datawere analyzed using HP CHEMSTATION™ software (Rev A08.03).

Capillary Zone Electrophophoresis (CZE)

The purity of Fab and F(ab′)₂ fragments was determined by CZE. Thisassay was run on a BIORAD BIOFOCUS™ 3000™ capillary electrophoresissystem with a BIOCAP XL™ capillary, 50 pm I.D., 44.6 cm total length and40 cm to the detector.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography was used to quantitate aggregates andfragments. This assay utilizes a TSK G3000 SWXL™, 7.8×300 mm column andruns on an HP 1100™ HPLC system. Samples were diluted to 10 mg/mL withthe mobile phase and injection volume was 20 μL. The mobile phase was100 mM K₂HPO₄ at pH 6.8 and the protein was eluted with an isocraticgradient at 0.5 m/min for 45 minutes. The eluent absorbance wasmonitored at 280 nm. Integration was done using HP CHEMSTATION™ software(Rev A08.03).

Biological Activity

The biological activity of Pertuzumab was determined by measuring itsability to inhibit proliferation of the human breast cancer cell lineMDA-MB-175-VII.

Example 1

Pertuzumab Fab and F(ab′)₂ antibody fragments were formulated at proteinconcentration of 1.0 mg/mL in the following buffer conditions:

-   -   10 mM citrate, 140 mM NaCl, pH 4.0;    -   10 mM succinate, 140 mM NaCl, pH 5.0;    -   10 mM succinate, 140 mM NaCl, pH 6.0;    -   10 mM histidine, 140 mM NaCl, pH 7.0; and    -   10 mM glycylglycine, 140 mM NaCl, pH 8.0.

Each formulation was filtered then aliquoted into 3 cc WHEATON™ USP TypeI glass vials sealed with TEFLON™ coated gray butyl stoppers. Sampleswere stored at 40±2° C. The stability analyses of drug product showedthat the Fab and F(ab′)₂ were most stable between pH 5.0 and 6.0.

TABLE 2 Effect of pH on degradation of Fab or F(ab′)₂ stored at 40° C.Fab F(ab′)₂ CZE SEC CZE SEC Formulation pH % Main Peak % Main Peak %Main Peak % Main Peak 4.0 74.1 96.7 43.6 89.4 5.0 83.2 96.4 65.4 94.06.0 82.9 96.2 69.0 92.3 7.0 83.9 96.4 62.3 91.3 8.0 72.7 96.4 49.2 89.8

Example 2

Pertuzumab was formulated into 20 mM histidine-acetate buffer with 120mM sucrose and 0.02% polysorbate 20. The pHs of formulations wereadjusted with acetic acid to final pH between 5.0 and 7.0. The proteinconcentration was 30 mg/mL. Each formulation was filled into 3 cc USPType I glass vials and stored at 40° C. for stability analysis. Theresults showed that Pertuzumab was most stable around pH 6.0.

TABLE 3 Effect of pH on degradation of Pertuzumab stored at 40° C.Temperature Storage Time SEC IEX Formulation pH (° C.) (wks) % Monomer %Main Peak 5.0 40 2 99.4 57.4 5.5 40 2 99.4 59.2 6.0 40 2 99.4 60.6 6.540 2 99.3 60.5 7.0 40 2 99.1 54.0 5.0 40 4 97.3 48.1 5.5 40 4 99.1 50.56.0 40 4 99.1 53.3 6.5 40 4 99.0 52.3 7.0 40 4 98.6 42.3

Example 3

Pertuzumab formulations at protein concentration of 100 mg/mL wereprepared in the following excipients:

-   -   (1) 10 mM histidine-HCl, 240 mM sucrose, 0.02% polysorbate 20,        pH 6.0;    -   (2) 10 mM histidine-acetate, 240 mM sucrose, 0.02% polysorbate        20, pH 6.0;    -   (3) 10 mM histidine-phosphate, 240 mM sucrose, 0.02% polysorbate        20, pH 6.0;    -   (4) 10 mM histidine-sulfate, 240 mM sucrose, 0.02% polysorbate        20 at pH 6.0.

Each formulation was filled into 3 cc FORMA VITRUM™ USP Type I glassvial sealed with FLUROTEC™ faced butyl rubber stoppers. Samples werestored at 30° C. and 40° C. and stability was evaluated for quality(CAC) and purity (SEC, IEC). The stability results showed thatPertuzumab in histidine-phosphate buffer degraded much faster than inother histidine buffers upon storage at 40° C. (FIG. 8 and FIG. 9 ).

Example 4

Pertuzumab was concentrated by ultrafiltration/diafiltration to variousconcentrations in the following buffers:

-   -   (1) 20 mM histidine-acetate, pH 6.0;    -   (2) 10 mM histidine-HCl, pH 6.0, and    -   (3) 10 mM histidine-sulfate, pH 6.0.

The turbidity of each formulation was measured before the filtration.The results, as shown in FIG. 10 , demonstrated that Pertuzumab samplesformulated in histidine-acetate and histidine-HCl had less amounts ofinsoluble aggregates than those in histidine-sulfate buffer.

Example 5

Pertuzumab was formulated at 30 mg/mL in 20 mM histidine-acetate, 120 mMsucrose, 0.02% polysorbate 20, pH 6.0. Pertuzumab was filled in 316L andHASTELLOY™ Stainless steel miniature tanks. All samples were stored at−20° C. and 5° C. and evaluated for quality (CAC), purity (SEC, IEC) andstrength (UV-Vis). The stability analyses showed that Pertuzumab wasstable in this formulation upon storage at −20° C. and 5° C. for atleast 3 months. The chloride free formulation is compatible with 316Land HASTELLOY™ stainless Steel tank.

TABLE 4 Stability of Pertuzumab in Stainless Steel Tanks Temp Time UVSpec. SEC IEC Tanks (° C.) (Months) CAC (mg/mL) (% monomer) (% mainpeak) 0 Pass^(a) 29.0 99.8 67.9 316L −20 3 Pass 28.9 99.7 66.8 5 3 Pass28.7 99.7 66.8 HASTELLOY ™ −20 3 Pass 29.1 99.7 66.8 5 3 Pass 28.8 99.767.7 ^(a)Pass for Color, Appearance and Clarity: Clear to slightlyopalescent, colorless to pale yellow solution.

Example 6

Pertuzumab was formulated using tangential flow filtration (TFF). Thefinal formulation contains 20 mM histidine-acetate, 120 mM sucrose,0.02% polysorbate 20, pH 6.0 at protein concentration of 30 mg/mL.Samples were filled into a 20 Ml FORMA VITRUM™ USP Type I glass vial,capped with the 20 mm FLUROTEC™ faced butyl rubber stoppers, and sealedwith aluminium flip-top caps. All samples were stored at −70° C., 5° C.,15° C., and stability was evaluated for quality (CAC), purity (SEC,IEC), strength (UV-Vis), and potency (Bioassay). The results showed thatPertuzumab is stable in this formulation upon storage at 5° C. and 15°C. for at least 3 months.

TABLE 5 Stability of Pertuzumab in glass vials SEC IEC Bioassay TempTime UV Spec. (% (% main (% specific (° C.) (Months) CAC (mg/mL)monomer) peak) activity) 0 Pass 29.2 99.8 64.1 83 −70 1 Pass 29.7 99.865.2 92 3 Pass 30.7 99.8 67.0 93 5 3 Pass 30.4 99.7 67.2 90 15 1 Pass29.7 99.7 64.4 78 3 Pass 30.4 99.7 65.5 93

Example 7

Pertuzumab was formulated at 100 mg/mL in the following bufferconditions:

-   -   (1) 10 mM histidine-HCl, pH 6.0;    -   (2) 10 mM histidine-HCl, 240 mM sucrose, pH 6.0;    -   (3) 20 mM succinate at pH 6.0; and    -   (4) 20 mM succinate, 240 mM sucrose at pH 6.0.

Each formulation was added with different concentration of polysorbate20. All samples were filled into 3 cc USP Type I glass vials and wereagitated horizontally at 70 rpm at room temperature for up to 7 days.The stability of each sample was evaluated at 7 day time point forturbidity. The results demonstrated that the use of polysorbate 20 inthe final formulation effectively prevented formation of insolubleaggregates. See FIG. 11 .

Example 8

Pertuzumab was prepared in the following formulations:

-   -   (1) 25 mg/mL Pertuzumab, 10 mM histidine-HCl, 240 mM sucrose, pH        6.0;    -   (2) 50 mg/mL Pertuzumab, 10 mM histidine-HCl, 240 mM sucrose, pH        6.0;    -   (3) 60 mg/mL Pertuzumab, 20 mM histidine-acetate, 120 mM        sucrose, pH 6.0.

Various amounts of polysorbate 20 were added to each formulation. Allsamples were filled into 3 cc USP Type I glass vials, and agitatedhorizontally at 70 rpm at room temperature for up to 7 days. Thephysical stability of each sample was evaluated at 7 day time point forturbidity. The results demonstrated that the use of polysorbate 20 inhistidine-HCl and sucrose formulation effectively prevented formation ofinsoluble particulates. The formulation containing histidine-acetate andsucrose appeared to have the same protective effect on protein aspolysorbate 20. See FIG. 12 .

Example 9

Pertuzumab was formulated as follows:

-   -   (1) 100 mg/mL protein, 10 mM histidine-HCl, pH 6.0;    -   (2) 100 mg/mL protein, 20 mM succinate, pH 6.0;    -   (3) 60 mg/mL protein, 20 mM histidine-acetate, pH 6.0.

Each formulation was mixed with different amounts of sucrose. Allsamples were sterilely filled into 3 cc USP Type I glass vials. Theywere then frozen at −70° C. and thawed at 5° C. three times. Thephysical stability of each sample was determined after the three cyclesof freezing and thawing. The results demonstrated that sucrose preventssoluble aggregate formation during the freeze-thawing process. See FIG.13 .

Example 10

The preferred Pertuzumab formulation for therapeutic use consistsessentially of 30 mg/mL Pertuzumab in 20 mM histidine acetate, 120 mMsucrose, 0.02% polysorbate 20, at pH 6.0.

Compound Concentration Amount/L Pertuzumab 30 mg/mL 30 g L-Histidine 20mM 3.10 g MW = 155.16 g/mol Glacial Acetic Acid 11.6 mM 0.66 mL MW =60.05 g/mol Density = 1.05 g/cm³ Sucrose 120 mM 41.1 g MW = 342.3 g/molPolysorbate 20 0.02% (w/v) 0.2 mL Density = 1.012 g/cm³ MW: Molecularweight420 mg dose vial configuration:Vial: 20 cc Formal Vitrum Type I glassStopper: 20 mm DAIKYO GREY™, fluoro-resin laminatedCap: 20 mm flip top aluminumFill volume: 14.50 mLDelivery: 14.0 mL Pertuzumab in normal saline IV bag.1050 mg dose vial configuration:Vial: 50 cc Formal Vitrum Type I glassStopper: 20 mm DAIKYO GREY™, fluoro-resin laminatedCap: 20 mm flip top aluminumFill volume: 36.0 mLDelivery: 35.0 mL Pertuzumab in normal saline IV bag.

Example 11

This example concerns another Pertuzumab formulation which has been usedin Phase I and Phase II clinical trials. The composition consists of 25mg/ml Pertuzumab, 10 mM Histidine-HCl buffer, 240 mM sucrose, 0.02%Polysorbate 20, pH 6.0.

Ingredient Concentration Pertuzumab 25 mg/ml L-His HCl•H₂O (MW 209.6)1.12 mg/ml (0.0125M) L-His(MW 155.2) 0.72 mgml (0.0099M) Sucrose(MW342.3) 82.15 mg/ml (0.240M) Polysorbate 20 0.2 mg/ml (0.02%)

Example 12

Cellular apoptosis is mediated by intrinsic and extrinsic pathways.Chemotherapy can cause cell damage and may trigger apoptosis by theintrinsic pathway in response to cellular damage. However, cancer cellsoften develop resistance to chemotherapy through mutations in the p53tumor suppressor gene (Ashkenazi A. Targeting Death and Decoy Receptorsof the Tumour-Necrosis Factor Superfamily. Nature Reviews 2:420-430(2002)). Death receptors, such as DR4 and DR5, located on the surface ofcells trigger apoptosis via the extrinsic pathway that does not involvep53. Agonistic molecules, such as Apo2L, bind to DR4 and DR5 receptorsand activate caspases 8 and 10 through Fas-associated death domain.Caspase 8 and 10 then activate caspases 3, 6, and 7 to induce apoptosis.Molecular signaling of death receptors on tumor cells has therapeuticpotential for the elimination of cancer cells that are resistant toconventional therapies and molecules, like Apo2L, are currentlyundergoing clinical evaluation.

“Apomab” is a full-length CHO derived humanized IgG1 constructed with alamda light chain. It is an agonist antibody against DR5 that has beenshown to induce apoptosis of various cancer cell lines. Preclinicalstudies using a murine tumor implant model have shown that Apomab hassimilar or improved tumor reduction compared to Apo2L. Apomab is beingevaluated as an anti-cancer agent in the indications of advanced solidtumors and Non-Hodgkin's Lymphoma (NHL). The heavy and light chain aminoacid sequences of Apomab used in these experiments are shown in FIGS. 27and 28 .

Preparation of Antibody Formulations

Recombinantly produced Apomab had very dilute protein concentration andhigh pH. The material was concentrated to approximately 20 mg/mL andexchanged into 20 mM sodium acetate, pH 5.0 buffer using a MilliporeLabscale tangential flow filtration (TFF) system with MILLIPOREPELLICON™ XL, PLCGC10, 50 cm membrane. Apomab samples were formulatedinto various buffer systems covering pH range from 4.0 to 7.0 usingsodium acetate, histidine acetate, and sodium phosphate withouttrehalose and TWEEN 20® using dialysis with a 10,000 Da molecular weightcut off membrane (Pierce, Inc). Trehalose at 240 mM was added in thelast dialysis. After dialysis, 0.02% TWEEN 20™ was added to theformulation and the samples were filtered with 0.22 pm filters(Millipore, Inc.). A 0.5 mL volume of Apomab was filled into sterile 3cc glass vials (Forma Vitrum, Inc.) and sealed with 13 mm stoppers(Daikyo, Inc). Protein stability was evaluated at −70° C., 5° C., 30°C., and 40° C. with storage for up to 3 months.

Stability of Apomab Formulation

For drug product stability testing, Apomab formulated bulk filled into 5cc FORMA VITRUM® glass vials were formulated. Vials were filled with 5.5mL of formulated antibody, fitted with 20 mm DAIKYO® stoppers, andstored at −70° C., 5° C., 30° C., and 40° C. in the upright position.

For drug substance stability testing, Apomab formulated bulk was sterilefiltered through a 0.22 μm filter and 10 mL was filled into autoclaved20 cc 316L stainless steel mini-tanks. The tanks were placed upright at−20° C. and 5° C. A 1 mL aliquot was aseptically removed from themini-tanks at specified time intervals to assess protein quality. Thecontrol vials were 1 mL aliquots in 3 cc glass vials stored at −20° C.

Color, Appearance, and Clarity

The clarity, appearance, and color of the samples were visually assessedunder white fluorescent light using a light inspection station withblack and white background. For analysis of the drug substance,mini-tank samples were transferred to a 3 cc glass vial for inspection.

pH

pH was measured at room temperature with THERMO ORION SURE-FLOW ROSS™semi-micro pH electrode for measuring buffers or THERMO ORION GLS™combination micro pH electrode for measuring protein pH screeningsamples, a Beckman microelectrode probe for Toxicology stabilitysamples. The METERLAB™ pHM240 pH/Ion meter (Radiometer Analytical) wascalibrated every day with buffer standards (EM Science) at pH 7 and pH4.

Concentration

Protein concentration was determined by ultraviolet absorptionspectroscopy using an AGILENT 8453™ spectrophotometer. The samples werediluted with appropriate formulation buffer blanks to give an absorbancefrom 0.5 to 1.0. The instrument was blanked with the diluent solutionand the spectrum was scanned from 240 to 500 nm. The absorbance value at320 nm was subtracted from the absorbance at 279 nm to correct foroffset and light scattering. The protein concentrations were calculatedby the following equation:

${{Conc}.\left( {{mg}/{mL}} \right)} = \frac{\left( {{A279} - {A320}} \right) \times {dilution}{factor}}{{absorptivity}{coefficient}{in}{{cm}^{- 1}\left( {{mg}/{mL}} \right)}^{- 1}}$

The absorptivity coefficient based on sequence was initially determinedto be 1.32 cm⁻¹ (mg/mL)⁻¹ and this value was used for the pH screeningstudies. A later value of 1.7 cm⁻¹ (mg/mL)⁻¹ was determined by aminoacid analysis and proteolysis methods and this value was used for thestability analysis of Apomab used in Toxicology studies.

Ion-Exchange Chromatography

Ion exchange chromatography was carried out on an 1100 series HPLC(Agilent Technologies, Inc.) equipped with a diode array detector.Chromatography was carried out on a PROPAC WCX-10™ (Dionex) column(4×250 mm) at a flow rate of 0.5 m/min and with column temperature at40° C. Mobile phase A was 25 mM sodium phosphate, pH 6.5. Mobile phase Bwas 100 mM sodium chloride in the same buffer as mobile phase A. Thecolumn was equilibrated with 100% mobile phase A. For pH screeningsamples an amount of 20 mg of Apomab was loaded onto the column and theabsorbance was monitored at 214 nm. Protein was eluted from the columnwith the following gradient:

Time (min) % A % B 0 100 0 50 0 100 51 100 0 70 100 0

For stability analysis of material used in the Toxicology studies anamount of 30 mg of Apomab was loaded onto the column and the absorbancewas monitored at 280 nm. Protein was eluted from the column with thefollowing gradient:

Gradient:

Time (min) % A % B 0 100 0 40.0 40 60 41.0 0 100 45.0 0 100 45.1 100 060.0 100 0

Size-Exclusion Chromatography

Size exclusion chromatography was carried out on an 1100 series HPLC(Agilent Technologies, Inc.) equipped with a diode array detector. Anamount of 50 μg Apomab was loaded onto a TSK Gel 3000SWXL™ (7.8×300 mm)column and run at a flow rate of 0.9 m/min for 20 minutes for pHscreening samples and 0.5 m/min for 30 minutes for Toxicology stabilitysamples with 0.20 M potassium phosphate, 0.25 M potassium chloride, pH6.2 as a mobile phase. Absorbance was monitored at 280 nm.

Potency

The purpose of the potency bioassay was to measure the ability of Apomabto kill Colo205 cells using ALAMARBLUE™. Colo205 is a colon carcinomacell line, which expresses both DR5 and DR4 death receptors. This assayincorporates a fluorometric/colorimetric growth indicator based ondetection of metabolic activity. ALAMARBLUE™ is a redox dye that is blueand non-fluorescent in oxidized state. The intracellular metabolicreduction converts it into a red color that is also fluorescent. Thechanges in color and fluorescence are proportional to the metabolicactivity and number of living cells. The signal decreased when cellsdie. Apomab was diluted in medium with anti-Fc and then Colo 205 cellswere added to Apomab samples and incubate at 37° C. for 48 hours.ALAMARBLUE™ is added for the last 2-3 hours. The plate was read at 530nm excitation and 590 nm emission to get relative fluorescence units(RFU). The data were analyzed by KALEIDAGRAPH™. A dilution curve ofkilling was generated.

Results Formulation pH Screen Study

The effect of pH on antibody stability was studied using Apomab producedfrom an unamplified stable cell line. For this analysis, Apomab wasformulated at 20 mg/mL antibody in 20 mM sodium acetate buffer at pH4.0, 4.5, 5.0, 5.5; 20 mM histidine acetate buffer at pH 6.0 and 6.5;and 20 mM sodium phosphate buffer at pH 7.0. All of the formulationscontained 240 mM trehalose and 0.02% TWEEN 20®. The formulations werestored for up to 3 months at temperatures of −70° C., 5° C., 30° C., and40° C. and protein stability was determined by various analyticalassays, including CAC, pH, concentration, SEC and IEC. No significantchanges in CAC, pH or protein concentration were observed during storageof the samples.

Analysis of the samples by SEC showed that no significant changesoccurred during storage at 5° C. and -70° C. However, degradationobserved as the formation of antibody fragments and soluble aggregatesoccurred during storage at 30° C. and 40° C. (FIG. 20 ). To compare theformulations, antibody monomer kinetics during storage was monitored andthe first-order rate constants were calculated. The obtained pH rateprofile for the loss in antibody monomer is shown in FIG. 21 . Theoptimal condition for the stability of antibody monomer was obtained byformulating in histidine acetate buffer at pH 6.0.

Apomab charge heterogeneity was monitored by IEC. No significant changesin the IEC profile occurred during storage at 5° C. and −70° C. However,degradation observed as the formation of acidic or basic variantsoccurred depending on the formulation (FIG. 22 ). In general, increasedbasic variants were formed at lower formulation pH and more acidicvariants were formed at higher formulation pH. To compare theformulations, IEC main peak kinetics was monitored during storage andthe first-order rate constants were calculated. The obtained pH rateprofile for the loss in IEC main peak is shown in FIG. 23 . The rateconstants observed by IEC were approximately 10 fold higher than thosefrom SEC (FIG. 21 ). Therefore, the loss in IEC main peak was theprimary degradation of the antibody that will ultimately limit theproduct shelf life. Furthermore, as observed by SEC, optimal antibodystability to stabilize IEC main peak was obtained by formulating inhistidine acetate buffer at pH 6.0.

Following the analysis of pH screening data described above, an Apomabformulation was selected that comprised 20 mg/mL antibody in 20 mMhistidine acetate, 240 mM trehalose, 0.02% polysorbate 20, pH 6.0. Forthe drug product, the vial configuration consisted of 5.5 mL fill in a 5cc FORMA VITRUM™ vial with a 20 mM DAIKYO™ West stopper. Apomab wasstored in stainless steel tanks.

The stability of Apomab Drug Product was evaluated in the 5 cc glassvial configuration described above. Vials were stored at −70° C.(controls), 5° C., 30° C., and 40° C. Samples were pulled at specifictime intervals and analyzed by the following assays: color, appearance,clarity (CAC), pH, protein concentration, SEC, IEC, and potency. Theresults from these assays are shown in Table 6 for samples stored at−70° C. and 5° C. and Table 7 for samples stored at 30° C. and 40° C.

TABLE 6 Stability Data for Apomab Stored at −70° C. and 5° C. Temp TimeSEC IEC Potency (° C.) Point Concentration (% (% main (% SpecificAcceptance Clarity Color pH (mg/mL) monomer) peak) Activity) Criteria:Report Report 6.0 ± 0.3 20 ± 2 ≥95% Report 60-140% NA T = 0 ClearColorless 5.9 20.2 99.8 63 94 −70 1 month Clear Colorless 6.0 20.5 99.863 86 −70 2 month Clear Colorless 6.0 20.4 99.7 64 91 −70 3 month ClearColorless 6.0 20.5 99.7 63 83 −70 6 month Clear Colorless 6.0 20.4 99.764 85 −70 9 month Clear Colorless 6.0 20.4 99.8 65 89 −70 12 month ClearColorless 6.0 20.8 99.7 63 107 5 1 month Clear Colorless 6.0 20.5 99.763 89 5 2 month Clear Colorless 6.0 20.4 99.7 64 99 5 3 month ClearColorless 6.0 20.6 99.7 63 84 5 6 month Clear Colorless 6.0 20.5 99.7 6493 5 9 month Clear Colorless 6.0 20.6 99.7 64 88 5 12 month ClearColorless 6.0 20.7 99.6 64 106

TABLE 7 Stability Data for Apomab Stored at 30° C. and 40° C. SEC IECPotency Temp (° C.) Time Point Concentration (% (% main (% SpecificAcceptance Clarity Color pH (mg/mL) monomer) peak) Activity) Criteria:Report Report 6.0 ± 0.3 20 ± 2 ≥95% Report 60-140% 30 1 month ClearColorless 6.0 20.6 98.2 59 91 30 2 month Clear Colorless 6.0 20.3 97.454 80 30 3 month Clear Colorless 6.0 20.6 97.2 49 74 30 6 month ClearColorless 6.0 20.2 94.1 37 51 30 9 month Clear Slightly 6.0 20.4 93.2 3155 yellow 30 12 month Clear Slightly 6.0 20.6 91.6 25 59 yellow 40 1month Clear Colorless 6.0 20.4 96.6 44 79 40 2 month Clear Colorless 6.020.0 93.7 31 64 40 3 month Clear Slightly 5.9 20.3 91.5 22 53 yellow 406 month Clear Slightly 6.0 20.2 83.9 NT 26 yellow 40 9 month ClearYellow 5.9 20.3 78.8 NT 25 40 12 month Clear Yellow 5.9 20.5 71.4 NT 31NT = not quantitated

No change in protein quality was observed after twelve months storage at−70° C. and 5° C. For instance, the pH remained at 6.0±0.3, Apomabappeared as a clear and colorless liquid, the protein concentrationremained at 20.0±2.0 mg/mL, and % monomer was unchanged. Furthermore,there was no significant change in % IEC main peak and % specificactivity determined by the cell-killing potency assay was within theassay precision of 60% to 140% specific activity. The results showedthat Apomab stored in 5 cc glass vials was stable for at least 12 monthsat 5° C.

Table 7 shows that changes in protein quality occurred at 30° C. and 40°C. SEC showed a decrease in % monomer with a rise primarily in fragmentspecies. Aggregates increase as well at higher temperature, but the ratewas much slower. However, the aggregates increase significantly after 6months at 40° C. IEC % main peak decreased with a corresponding increasein acidic variants. Basic peaks decreased slightly after 2 months at 40°C. and 9 months at 30° C. After six months of storage at 40° C.,degradation occurred to an extent that IEC main peak could no longer beintegrated. The cell killing bioassay showed loss of % specific activityat higher temperature with longer storage time. Protein concentrationand pH were unchanged. The solution becomes slightly yellow after 3months at 40° C. and 9 months at 30° C. and becomes yellow after 9months at 40° C.

Drug Substance Stability

Freeze-thaw stability data for drug substance are shown in Table 8.

TABLE 8 Freeze-Thaw Stability Data for Apomab Filled in MiniatureStainless Steel Tanks Temp (° C.) Freeze-Thaw Concentration SEC(Frozen/thaw) Cycle No. Clarity Color pH (mg/mL) (% Monomer) AcceptanceCriteria: Report Report 6.0 ± 0.3 20.0 ± 2.0 ≥95% Control 0 ClearColorless 6.0 20.9 99.6 (unfrozen) −20/25 1 Clear Colorless 6.0 20.899.6 −20/25 2 Clear Colorless 6.0 20.8 99.6 −20/25 3 Clear Colorless 6.020.9 99.6

TABLE 9 Stability Data for Apomab Filled in Miniature Stainless SteelTanks SEC IEC Potency Time Concentration (% (% main (% Specific Temp (°C.) Point Clarity Color pH (mg/mL) monomer) peak) Activity) AcceptanceCriteria: Report Report 6.0 ± 0.3 20 ± 2 ≥95% Report 60-140% NA T = 0Clear Colorless 5.9 20.0 99.7 63 88 −20 1 month Clear Colorless 6.0 20.699.7 63 107 −20 3 month Clear Colorless 6.0 20.6 99.7 63 82 −20 6 monthClear Colorless 6.0 20.3 99.7 64 92 −20 9 month Clear Colorless 6.0 20.699.7 64 92 −20 12 month Clear Colorless 6.0 21.2 99.7 65 94 5 1 monthClear Colorless 6.0 20.5 99.7 62 95 5 3 month Clear Colorless 6.0 20.799.6 62 71 5 6 month Clear Colorless 6.0 20.4 99.5 62 84 5 9 month ClearColorless 6.0 20.8 99.4 61 84 5 12 month Clear Colorless 6.0 21.3 99.259 82

No significant changes in the chemical characteristics of the proteinwere observed after being frozen at −20° C. for at least 15 hours andthawed at ambient temperature three times. For example, Apomab appearedas a clear and colorless liquid, the pH remained at 6.0±0.3, and the SECmonomer peak percentage was unchanged.

Apomab stability in stainless steel containers was evaluated at −20° C.and 5° C. (Table 9).

Samples were aseptically pulled from the mini-tanks at specificintervals and analyzed.

Apomab showed no change in protein quality at 5° C. by pH, CAC, proteinconcentration and % main peak by IEC but lost 0.1% monomer by SEC every3 months. Decreased potency was observed during storage at 5° C. for 3months. However, the potency of the sample increased again at the 6 and9 month timepoints. Therefore, the observed potency difference at the 3month timepoint was attributed to assay variation. Apomab showed nochange in protein quality at −20° C. by pH, CAC, protein concentration,% monomer by SEC, % main peak by IEC, and no significant change inpotency. The stability data show that Apomab is stable for at least 1year at −20° C. and three months at 5° C.

CONCLUSION

Formulation screening studies were performed to select a formulation forApomab. A pH screen covering the pH range 4.0 to 7.0 using sodiumacetate, histidine acetate, and sodium phosphate as buffers with 240 mMtrehalose dihydrate and 0.02% polysorbate 20 showed that Apomab is moststable in solution at pH 6.0. Therefore, a formulation consisting of 20mM histidine acetate, 240 mM trehalose, 0.02% polysorbate 2, pH 6.0 wasdeveloped and demonstrated experimentally to be stable. Using thisformulation, Apomab was shown to be stable for at least 12 months at 5°C. Furthermore, Apomab was shown to be stable for at least 12 months at−20° C. and three months at 5° C. when stored in 316L stainless steelcontainers. Apomab was also shown to be stable when subjected to up to 3freeze/thaw cycles.

1. A method for reducing deamidation or aggregation of a therapeuticmonoclonal antibody, comprising formulating the antibody in ahistidine-acetate buffer, pH 5.5 to 6.5.
 2. The method of claim 1comprising evaluating any antibody deamidation or aggregation before andafter the antibody is formulated.
 3. The method of claim 1 wherein theantibody binds to domain II of HER2.
 4. The method of claim 1 furthercomprising formulating the antibody in a saccharide and a surfactant. 5.The method of claim 3 wherein the antibody is formulated at aconcentration from 20 mg/mL to 40 mg/mL, the antibody comprises thevariable light and variable heavy amino acid sequences in SEQ ID Nos. 3and 4, respectively, the saccharide is sucrose which is formulated at aconcentration from about 60 mM to about 250 mM, and the surfactant ispolysorbate 20 which is formulated at a concentration from about 0.01%to about 0.1%.
 6. The method of claim 3 wherein the HER2 antibodycomprises a light chain amino acid sequence selected from SEQ ID No. 15and 23, and a heavy chain amino acid sequence selected from SEQ ID No.16 and
 24. 7. The method of claim 1 wherein the pH of the buffer is fromabout 5.8 to about 6.2.
 8. The method of claim 5 wherein the antibody isa full length antibody.
 9. The method of claim 1 wherein the formulationis aqueous and has not been subjected to prior lyophilization, and isadministered to a subject.
 10. The method of claim 9 wherein theformulation is administered to the subject intravenously.
 11. The methodof claim 1 wherein the antibody comprises Pertuzumab formulated in anamount from 20 mg/mL to 40 mg/mL.
 12. A formulation comprisingPertuzumab in a histidine-acetate buffer, pH 5.5 to 6.5.
 13. Theformulation of claim 12 further comprising saccharide and surfactant.14. The formulation of claim 12 wherein the Pertuzumab concentration inthe formulation is from 20 mg/mL to 40 mg/mL.
 15. The formulation ofclaim 13 comprising 60 mM to 250 mM sucrose.
 16. The formulation ofclaim 13 comprising 0.01% to 0.1% polysorbate.
 17. The formulation ofclaim 12 wherein the pH of the buffer is from 5.8 to 6.2.
 18. Theformulation of claim 12 which is aqueous and has not been subjected toprior lyophilization, and is administered to a subject.
 19. Theformulation of claim 18 which is administered to the subjectintravenously.